Bachelor-thesis Antinéa BABARIT

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ER-Import Defects in
Phosphorylation
Mutants of Sbh1p
BACHELOR THESIS (B.SC)
Submitted by Antinéa BABARIT
Human and Molecular Biology Center - Zentrum für Human- und
Molekularbiologie (ZHMB)
Faculty of Natural Sciences and Technology III and Faculty of
Medicine
Saarland University, Germany
Saarbrücken, July 2015

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The experimental work depicted in this thesis has been carried out at the department of Microbiology
at Saarland University, Germany.
First Reviewer: Prof. Dr. Karin Römisch
Second Reviewer: Joseph Schacherer

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Biology is the study of complicated things that have the
appearance of having been designed with a purpose.
- Richard Dawkins
Success consists of going from failure to failure without loss of
enthusiasm.
- Winston Churchill

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Table of contents
Table of contents............................................................................................................................4
List of Figures .................................................................................................................................7
List of Tables...................................................................................................................................8
I. Introduction ............................................................................................................................... 9
1. From translation to secretion: The Secretory Pathway...................................................... 9
1.1 - Targeting to the ER ...........................................................................................................9
1.2 - ER to Golgi trafficking.................................................................................................... 10
1.3 - Secretion and Exocyst...................................................................................................... 11
2. Across the membrane: Protein Translocation into the ER .............................................. 12
2.1 - The Sec61 complex ...........................................................................................................12
2.1.1 - The Sec61p Subunit .................................................................................................13
2.1.2 - The Sbh1p Subunit..................................................................................................13
2.1.3 - The Sss1p Subunit .................................................................................................. 14
2.2 - The Ssh1 Complex........................................................................................................... 14
2.3 - The Sec Complex .............................................................................................................15
3. Post-translational modifications: Processing in the ER....................................................15
3.1 - Evolving into glycoprotein...............................................................................................15
3.1.1 - N-linked glycosylation............................................................................................15
3.1.2 - Oligosaccharyl Transferase Complex .................................................................. 16
3.2 - More post-translational events..................................................................................... 16
3.3 - Protein inspection - ER Quality Control .......................................................................17
3.4 - The good, the bad and the misfolded - ER-Associated Degradation...........................17
3.5 - Stress in the ER and Unfolded Protein Response......................................................... 19
4. Aim of this study...................................................................................................................20
II. Materials and Methods ...........................................................................................................22
1. Materials .................................................................................................................................22
1.1 - Laboratory equipment, chemicals, reagents and consumables...................................22
1.2 - Media and Buffers...........................................................................................................28
1.3 - Bacterial and yeast strains.............................................................................................33
1.4 - Plasmids ..........................................................................................................................35

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1.5 - Antibodies........................................................................................................................36
1.6 - Softwares.........................................................................................................................37
2. Methods .................................................................................................................................37
2.1 - Sterilization.....................................................................................................................37
2.2 - Growth of S. cerevisiae...................................................................................................37
2.3 - Growth of E. coli.............................................................................................................37
2.4 - Plasmid Extraction.........................................................................................................37
2.5 - Transformation of S. cerevisiae.....................................................................................38
2.6 - Preparation of Cell Extracts..........................................................................................38
2.7 - Protein Gel Electrophoresis and Western Blot Analysis.............................................38
2.7.1 - Protein Gel Electrophoresis..................................................................................38
2.7.2 - Western Blot Analysis ..........................................................................................39
2.7.3 - Membrane stripping .............................................................................................39
2.8 - Cycloheximide Chase Experiments...............................................................................39
2.9 - Pulse Experiments..........................................................................................................39
2.9.1 - Pulse Labeling........................................................................................................39
2.9.2 - Immunoprecipitation.......................................................................................... 40
2.9.3 - Gel Electrophoresis and Revealing .................................................................... 40
2.10 - Native Immunoprecipitation ...................................................................................... 40
III. Results .....................................................................................................................................42
1. Import of Glucosidase I into the ER ....................................................................................42
1.1 - Phosphorylation- and double transmembrane mutants ..............................................42
1.2 - Combined mutants .........................................................................................................44
2. Oligosaccharyl Transferase Complex and Sbh1p.............................................................. 46
3. ER-Associated Degradation Defects ...................................................................................47
3.1 - Phosphorylation- and double transmembrane mutants .............................................47
3.2 - Combined mutants........................................................................................................ 49
4. Test on Antibodies Directed Against Sbh1p ..................................................................... 49
IV. Discussion ...............................................................................................................................52
1. Import of Glucosidase I into the ER ....................................................................................52
2. Oligosaccharyl Transferase Complex and Sbh1 .................................................................53

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3. ER-Associated Degradation Defects ...................................................................................54
4. Test on Antibodies Directed Against Sbh1p ......................................................................56
V. Conclusion................................................................................................................................57
VI. References...............................................................................................................................58
VII. Abbreviations ........................................................................................................................62
VIII. Annex....................................................................................................................................65
1. Auxotrophic amino-acids .....................................................................................................65
2. Supplementary figures......................................................................................................... 66
IX. Acknowledgements............................................................................................................... 68

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List of Figures
Figure 1 - The SRP targeting cycle. ............................................................................................. 10
Figure 2 - ER to Golgi trafficking.................................................................................................11
Figure 3 - Sec61 complex...............................................................................................................13
Figure 4 - Calnexin/Calreticulin cycle. ...................................................................................... 18
Figure 5 - ERAD Pathway. ...........................................................................................................20
Figure 6 - Import of Gls1 in SBH1 mutant strains. ....................................................................43
Figure 7 - Amount of Gls1 in the cell for different SBH1 mutants...........................................43
Figure 8 - Import of Gls1 in SBH1 mutant strains. ................................................................... 44
Figure 9 - Import of Gls1 in SBH1 combined mutant strains...................................................45
Figure 10 - Amount of Gls1 in the cell for different combined SBH1 mutants.......................45
Figure 11 - Import of CPY in SBH1 mutants. ............................................................................. 46
Figure 12 - Degradation of CPY* in SBH1 mutant strains over time. .................................... 48
Figure 13 - Degradation of CPY* in combined SBH1 mutant strains over time.....................50
Figure 14 -Testing of a new Sbh1 antibody and comparison with the previously used one..51
Figure 15 - Degradation of CPY* in SBH1 mutant strains over time...................................... 66
Figure 16 - Import of CPY in SBH1 mutant strains. ..................................................................67
Figure 17 - Import of CPY in SBH1 combined mutant strains. ................................................67

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List of Tables
Table 1 - Chemicals and reagents used in this study................................................................22
Table 2 - Laboratory equipment used in this study .................................................................25
Table 3 - Consumables used in this study .................................................................................27
Table 4 - Composition of the media used in this study...........................................................28
Table 5 - Composition of the solutions for yeast transformation...........................................29
Table 6 - Composition of the solutions for cell extract preparation ......................................30
Table 7 - Composition of the solutions for SDS-PAGE and Western Blot.............................30
Table 8 - Composition of the solutions for pulse experiments................................................31
Table 9 - Composition of the solutions for native immunoprecipitation..............................33
Table 10 - Escherichia coli (E.coli) strains used in this study..................................................33
Table 11 - S. cerevisiae strains used in this study ......................................................................34
Table 12 - Plasmids used in this study........................................................................................35
Table 13 - Antibodies used in this study ....................................................................................36
Table 14 - Softwares used in this study ......................................................................................37
Table 15 - Composition of Synthetic Complete amino acid drop-out mixture* for S.
cerevisiae.......................................................................................................................................65

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I. Introduction
1. FROM TRANSLATION TO SECRETION: THE SECRETORY
PATHWAY
Proteins are the basis element of all living cells. They have many roles in the cell life, such
as in structure (e.g. actine or collagene), in mobility (e.g. myosine), in DNA conditioning (e.g.
histones), in the catalysis of reactions (e.g. enzymes), etc. These molecules are needed in various
locations in and outside the cell, and they don’t come into being where they are needed (Alberts et
al., 2002).
In eukaryotes, after their biosynthesis, proteins have to follow a specific pathway that will
allow them to be correctly folded and modified, before they can be delivered where they belong.
This is called the secretory pathway. It’s a journey across the cell organelles that begins in the cytosol
(Alberts et al., 2002).
1.1 - Targeting to the ER
In 1999 Blobel won the Nobel Prize in Physiology and Medicine for discovering that proteins
have intrinsic signals that govern their transport and localization in the cell (Blobel and
Dobberstein, 1975). This signal is a small peptide of fifteen to thirty amino-acids, with a hydrophobic
core and a positive charge at its N-terminus and that is usually found at the N-terminus of secretory
proteins (Cooper and Hausman, 2006). A signal anchor also exists for tail anchored proteins, whose
charge distribution at the first transmembrane domain (TMD) determines the orientation of the
protein insertion (Borgese and Fasana, 2011).
Once this sequence emerges from the ribosome, there is a pause in the translation. The
signal recognition particle (SRP) binds to the signal peptide and forms a complex with the ribosome
(Figure 1). The signal sequence is then recognized by the 54-kDa polypeptide of the SRP. This
complex containing the nascent chain (NC), the ribosome and the SRP is then targeted to the
endoplasmic reticulum (ER) membrane, where it binds to the SRP receptor, consisting of an α and
a β subunit, next to a protein conducting channel, the Sec61 complex. The NC is inserted into this
translocon, and the ribosome binds to the channel. Guanosine Triphosphate (GTP) binds to the
GTP-binding G domain of the SRP and to both subunits of the SRP receptor, and its hydrolysis
allows the release of both the SRP and its receptor, that then begin a new targeting cycle (Rapoport,
1992). It sometimes happens that the SRP and its receptor are not enough to allow the translocation
of a protein. They can then use some help, such as the translocating chain-associating membrane
protein (TRAMp) for membrane insertion (Gruss et al., 1999).
The signal peptide is then removed while the NC is emerging into the ER lumen by an
enzyme, present on the lumenal side of the ER membrane, called signal peptidase. In the case of
membrane proteins, it is possible that there is a stop transfer signal, that arrests the translocation,
and the appropriate hydrophobic region migrates laterally into the membrane to become a TMD.
Once in the ER lumen, proteins are taken over by chaperone molecules, that prevent inappropriate
interactions between proteins, as well as protein aggregation, and promote protein folding (Vitale
and Denecke, 1999).
The SRP targeting is a co-translational way to cross the ER membrane, but it may happen
that proteins cannot be translocated co-translationally, because they are too small or their signal
sequence is not located at their N-terminus (e.g. tail-anchored (TA) proteins). In the case of C-tail-

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anchored proteins, such as the yeast protein Sbh1p, the only targeting determinant is present at
their C-terminus, and it is only available when the protein is released from the ribosome, thus these
proteins have to be translocated post-translationally. Some of the post-translational targeting
mechanisms have been discovered, and an example in yeast is the guided entry of tail-anchored
proteins complex (GET complex; Borgese and Fasana, 2011). Get3 is an Adenosine Triphosphatase
(ATPase) that binds specifically to the C-terminally localized hydrophobic domains on newly
synthesized proteins. Get1 and Get2 are two membrane proteins localized on the ER membrane that
serve as receptor, to which binds the Get3-TA complex. After the binding of this complex to the
receptors, the TA protein is inserted into the membrane and can be further routed to another cell
organelle when needed (Schuldiner et al., 2008).
Figure 1 - The SRP targeting cycle. This illustration shows the SRP targeting of nascent proteins to the ER
membrane. The signal sequence is first bound by the SRP (step 1), and the complex thereby formed binds to
the SRP receptor on the surface of the ER-membrane (step 2). The SRP is released by GTP hydrolysis and
starts a new cycle while the signal sequence is inserted into the translocon (step 3), where it is then cleaved
by SP (step 4), and the newly synthesized protein is finally released in the ER lumen (step 5) for further
modifications. Illustration from Cooper and Hausman, 2006.
1.2 - ER to Golgi trafficking
Once in the ER lumen, proteins can be subjected to several post-translational modifications,
such as N-glycosylation by the Oligosaccharyl Transferase complex, disulfide bond formation by the
protein disulfide isomerase (PDI) and addition of Glycosylphosphatidylinositol (GPI)-anchors.
Those modifications are necessary for the correct folding of proteins by chaperones (Cooper and
Hausman, 2006).
When proteins are fully folded and modified, they are incorporated into small transport
vesicles (Figure 2). A coated protein complex (COPII) initiates the budding of this vesicle from the
ER membrane, in association with other proteins. Those vesicles are released in the cytosol,

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proximal to the Golgi apparatus, and are transported to the ER-Golgi intermediate compartment
(ERGIC) from where they are led to the cis-Golgi by microtubules and dynein, in COPI-coated
vesicles. Some proteins need to be transported back to the ER, and this retrograde transport from
Golgi to the ER is mediated by COPI coated-vesicles and require microtubules and kinesin. (Lord et
al., 2013). Dynein and kinesin are motor proteins that hydrolyze ATP, allowing the anterograde
movement on the microtubule (in the case of kinesin) or the retrograde movement (in the case of
dynein) (Berg et al., 2002).
Once the COPII-coated vesicle is fused with the membrane of the cis-Golgi, a process named
cisternal migration begins. Proteins move through several compartments (cis cisternae, medial
cisternae and trans cisternae), where they may suffer additional post-translational modifications,
such as N-linked glycosylation, O-linked glycosylation and phosphorylation. They finally arrive in
the trans-Golgi network (TGN), in close proximity to the plasma membrane, where they are
destined to be secreted (Lodish et al., 2000). Membrane proteins follow the same pathway to reach
their final destination.
Figure 2 - ER to Golgi trafficking. This illustration represents the vesicle transport that occurs between the ER
and the Golgi. Vesicles bud from the ER membrane, coated by COPII, and travel to the ERGIC (intermediate
compartment), where they are then lead to the cis-Golgi by COPI coated vesicles (not shown). Proteins move
to the trans-Golgi, and then to the TGN to be secreted. Retrograde transport to the ER is mediated by COPI-
coated vesicles. Modified from Miller and Krijnse-Locker, 2008.
1.3 - Secretion and Exocyst
The proteins in the TGN are destined to be secreted. Those secretory proteins are sorted in
two types of vesicles, affecting the way they are secreted. In the case of proteins that have to be
continuously released from the cell, such as those that build the extracellular matrix, they are
transported into vesicles that directly move and fuse with the plasma membrane, allowing the
release of their content by exocytosis that is mediated by the exocyst complex. The other group

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contains proteins whose secretion has to be regulated, such as enzymes or hormones. In the TGN,
those proteins are sorted into secretory vesicles and stored inside the cell, waiting for a stimulus to
be secreted. This stimulus can be a neural or a hormonal signal, which leads to an increase of the
cytosolic Ca2+
concentration in the cytosol. This increase of Ca2+
triggers the fusion of the vesicle
membrane with the plasma membrane, and the release of the vesicle’s content (Lodish et al., 2000).
The exocyst is a 750-kDa complex, consisting in the following subunits: Sec3p, Sec5p, Sec6p,
Sec8p, Sec10p, Sec15p, Exo70p and Exo84p. The involvement of this complex in the exocytosis of
vesicles has first been identified in yeast (Lipschutz et al., 2003). Before the fusion with the plasma
membrane, vesicles containing the secretory proteins are recognized by the exocyst complex that
functions as a tethering factor for the vesicles at the plasma membrane (Toikkanen et al., 2003).
Sec4 is a small GTP-bound Rab protein found on the cytosolic side of the secretory vesicles’
membrane that interacts with the Sec15p subunit of the exocyst complex, presumably for specific
secretory vesicle recognition (Hutagalung and Novick, 2011; Lipschutz and Mostov, 2002). The
exocyst may form an initial connection between vesicle and plasma membrane, bringing the vesicle
close enough to promote SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein
receptor) complex formation and vesicle fusion and/or play a role in regulating SNARE assembly
(Heider and Munson, 2012).It has also been shown that the β subunit of the Sec61 translocon
interacts with the exocyst although they are located at opposite ends of the secretory pathway
(Toikkanen et al., 2003).
2. ACROSS THE MEMBRANE: PROTEIN TRANSLOCATION INTO
THE ER
As described before, proteins can be translocated into the ER co- or post-translationally,
according to their length and to the localization of their targeting sequence.
Several translocation complexes exist within the ER membrane, which are activated
depending on which type of translocation will be used. The three main ones are the Sec61 complex,
the Ssh1 complex and the Sec complex (Zimmermann et al., 2011).
2.1 - The Sec61 complex
The Sec61 complex is a protein translocation complex that is found in the ER membrane in
eukaryotic cells. It has a homologue in bacteria and archea, named SecYEG, what shows that this
complex is evolutionary conserved (Osbourne et al., 2005; Van den Berg et al., 2004; Wilkinson et
al., 1996). It consists of three subunits. Sec61α, Sec61β and Sec61γ in mammals; Sec61p, Sbh1p and
Sss1p in yeast (Figure 3); SecY, SecG and SecE in bacteria and archea. It is involved in co-translational
protein translocation into the ER, and post-translational protein import when it is part of the
heptameric Sec complex, forming the protein conducting channel (Finke et al., 1996; Harada et al.,
2011; Kalies et al., 1994). This complex also plays a role in the ER-Associated Degradation (ERAD)
pathway, as part of a large and dynamic complex consisting of an ER-resident ubiquitin ligase
(Hrd1), the Sec61 channel, Sec63p and the proteasome 19S regulatory particle (RP) (Römisch, 2005).

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In addition to being able to translocate proteins through the ER membrane, the Sec61 complex also
opens laterally, within the membrane, to allow the integration of hydrophobic transmembrane (TM)
segments of a membrane protein (Osbourne et al., 2005).
Figure 3 - Sec61 complex. This figure represents the Sec61 complex in yeasts, consisting of three subunits:
Sec61p, that forms the core of the channel, Sbh1p and Sss1p.
2.1.1 - The Sec61p Subunit
Sec61p is the main subunit of the Sec61 complex. Sec61p forms an aqueous channel whose
opening and closing is tightly regulated (Mothes et al., 1994; Pilon et al., 1998). It is a protein that
spans the membrane ten times, with its N- and C-termini in the cytosol. Its structure is closed by a
lumenal plug domain and a hydrophobic constriction ring (Junne et al., 2010). These are proposed
to prevent or reduce ion permeability during protein translocation (Gogala et al., 2014).
It is essential for the survival of the cell and with Sss1p, it forms the core of the translocation
channel (Osbourne et al., 2005). It possesses two larges loops, L6 and L8, on the cytoplasmic side of
the ER, and a large one, L7, on the lumenal side. L6 and L8 are important for ribosome binding
during co-translational import and L7 is proposed to be interacting with lumenal chaperones and/or
misfolded protein complexes in order to open the channel for the export of proteins for degradation
in the cytosol (Tretter et al., 2013). The N-terminal region also seems to be particularly important to
the function of this protein (Pilon et al., 1998).
2.1.2 - The Sbh1p Subunit
Sbh1p is a small 82 a.a. C-tail-anchored protein in the ER membrane with largely
unstructured cytosolic domain and single α-helical TMD consisting of 25 a.a. and with 7 a.a. more
presumably protruding into the lumen of the ER (Feng et al., 2007).
This protein is not essential to the survival of the cell. Nevertheless, the deletion of both
SBH1 and SBH2 (its homologue that encodes a protein in the Ssh1 complex) leads to high
temperature sensitivity (Soromani et al., 2012). It was also discovered that co-translational protein
translocation is reduced in this mutant and that it manifests N-glycan trimming defects at
permissive temperatures (Feng et al., 2007).
The cytosolic domain of Sbh1p can be phosphorylated, and Soromani et al. (2012) discovered
that several phosphorylation sites might exist. Among the six phosphorylation sites suggested by
bioinformatics tools, it turned out that their substitution with non-phosphorylable a.a., even in
combination with the other, did not affect in any manner the function of Sbh1p. However, they
discovered a new phosphor-acceptor site, a Thr at position 5, by mass spectrometry, and the further
investigation on this site displayed interesting results. In fact, it seems that even when this site is
mutated, the protein is not hypophosphorylated, suggesting that another substrate can be used by
the same kinase, such as the Ser at position 3. This phosphorylation site at position T5, as well as

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the two Pro on its side evolved independently, in Saccharomyces cerevisiae (S. cerevisiae) and in
mammals, but are not found in other yeasts, in other vertebrates, and in unicellular eukaryotes,
suggesting that this site might be relevant for its function in the protein translocation, since this
phosphorylation only takes place when the subunit is associated in the Sec61 complex.
The TMD of Sbh1p was also demonstrated as being an important domain of the protein.
Feng et al. (2007) showed that the expression of the TMD alone in strains lacking both SBH1 and
SBH2 was enough to rescue the temperature sensitivity. Moreover, it was proven to be sufficient to
ensure Sbh1p functions, and its interaction with other partners, such as the SP, the ribosome (Feng
et al., 2007) and the exocyst (Toikkanen et al., 2003).
These data indicate that Sbh1p acquired function independent of protein translocation into
the ER or regulatory function that coordinate translocation with other events (Feng et al., 2007).
Since this subunit is not essential, it was suggested that its function may be to enhance the
speed of the translocation or the efficiency of the targeting (Soromani et al., 2012), but this process
has been demonstrated so far.
2.1.3 - The Sss1p Subunit
Sss1p is a 9-kDa TA protein that together with the Sec61p subunit, forms the core of the Sec61
complex, and thus is essential for cell viability (Falcone et al., 2011).
The C-terminal end of the hydrophobic sequence of this protein plays a role in the proper
assembly of the Sec61 and Sec complexes, as it was shown that mutations in it compromises the
stability of these complexes, resulting in a severe defect in the function of the Sec61 complex and
leading to a complete reorganization of the ER structure (Falcone et al., 2011). Moreover, its
cytoplasmic region has been shown to interact with the OST, like the TMD of Sbh1p (Chavan et al.,
2005).
The precise mechanism of action of this protein is largely unknown, but several hypotheses
have been proposed. It may help to maintain the membrane permeability barrier, facilitate the
oligomerization of the Sec61 complex and it may clamp together the two halves of the Sec61p subunit
(Falcone et al., 2011).
2.2 - The Ssh1 Complex
The Ssh1 complex is a trimeric complex that is structurally related to the Sec61 complex. It
consists of three subunits: Ssh1p, Sss1p and Sbh2p (a homologue of Sbh1p) (Finke et al., 1996). It is
involved exclusively in co-translational translocation in the ER (Soromani et al., 2012).
It has also been proposed that it is involved in the ERAD pathway (Feng et al., 2007). As the
Sec61 complex, the Ssh1 complex also interacts with membrane bounds ribosomes, but does not
associate with the Sec62/63p complex to form a heptameric complex (Finke et al., 1996).
This complex is not essential for cell viability, but it is required for normal growth rates. It
is found in equal abundance as the Sec61 complex in yeast microsomes, which is the reason why it
is thought that both can transport distinct proteins, or that two different targeting pathways are
used. An alternative hypothesis is that having two co-translational translocation systems could
allow the cell to regulate co- and post-translational pathways independently from each other (Finke
et al., 1996).

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2.3 - The Sec Complex
The Sec complex is a heptameric complex that mediates post-translational import into the
ER of proteins with moderately hydrophobic signal sequences in yeast. It consists of the association
between the tetrameric Sec62/Sec63 complex (containing Sec63p, Sec62p, Sec71p and Sec72p) and
the Sec61 complex (Deshaies et al., 1991; Osbourne et al., 2005). The energy required for post-
translational translocation comes from the hydrolysis of ATP by the Kar2p chaperone molecule in
the ER lumen, a homologue of the mammalian BiP (Pilon et al., 1998).
Proteins using the post-translational pathway are kept in a translocation competent
conformation by molecular chaperones, which also play a role in the targeting to the Sec complex
(Falcone et al., 2011). The Sec62/63 complex plays a crucial role in targeting of SRP-independent
protein substrates to the protein conducting channel. It is also proposed to play an important role
in the assembly of the post-translocon (Harada et al., 2011).
3. POST-TRANSLATIONAL MODIFICATIONS: PROCESSING IN
THE ER
Since their first a.a. emerge in the ER lumen, proteins are subject to several events, that will
prepare them for their journey through the secretory pathway.
The proteins first undergo some modifications (N-linked glycosylation, disulfide bond
formation, addition of GPI-anchors, etc). Their folding is then controlled by a process called ER
Quality Control (ERQC) and they are sent to further organelles or retained in the ER to be led to
the cytosol for degradation. The ER-Associated Degradation pathway sends the misfolded or
unfolded proteins into the cytosol for degradation by the 26S proteasome, but when it is not
efficient, it leads to stress in the ER, and to a specific response called Unfolded Protein Response
(UPR; Lodish et al., 2000).
3.1 - Evolving into glycoprotein
3.1.1 - N-linked glycosylation
As soon as a glycosylatable site in the NC emerges in the ER lumen, an oligosaccharide is
transferred to the NH2 group of an asparagine in a selected Asparagine-X-Serine/Threonine (Asn-
X-Ser/Thr) sequence, with X being any a.a. except Proline (Pro; Aebi, 2013).
The precursor oligosaccharide is formed by a fourteen sugar long chain constisting of two
residues of N-acetylglucosamine (GlcNAc), nine residues of mannose (Man) and three residues of
glucose (Glc) and is anchored in the ER membrane by a lipid molecule called dolichol diphosphate
(Dol-P-P; Herscovics and Orlean, 1993). This oligosaccharide is transferred to the target Asn in a
single enzymatic step catalyzed by membrane bound enzyme called Oligosaccharyl Transferase,
which has its active site exposed to the lumenal side of the ER membrane (Silberstein and Gilmore,
1996).
Since most of the proteins are imported in a co-translational way into the ER, N-linked
oligosaccharide are almost always added during protein biosynthesis (Alberts et al., 2002).

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Although N-linked glycosylation is the most common type of glycosylation, oligosaccharides
can be linked to the hydroxyl group on the side chain of a serine, threonine or hydroxylysine (Hyl)
a.a. This process is called O-linked glycosylation and happens in the Golgi (Alberts et al., 2002).
3.1.2 - Oligosaccharyl Transferase Complex
The Oligosaccharyl Transferase Complex (OST) consists of the following subunits: Ost1p,
Ost2p, Stt3p, Wbp1p and Swp1p, which are encoded by essential genes, and Ost3p, Ost4p, Ost5p
and Ost6p, which are non-essential, but required for optimal activity of the enzyme (Chavan and
Lennarz, 2006; Silberstein and Gilmore, 1996).
In yeast, two isoforms of this complex have been found. One is made of eight subunits,
including Ost3p, but not Ost6p, and the other contains Ost6p, but not Ost3p. It has been shown
that those two complexes interact with two different translocation complexes: Ost3p interacts
specifically with the Sbh1p subunit of the Sec61 complex, and Ost6p interacts specifically with the
Sbh2p subunit of the Ssh1 complex (Yan and Lennarz, 2005). A severe underglycosylation phenotype
in soluble and membrane proteins has also been identified for mutants lacking both OST3 and OST6
(Chavan and Lennarz, 2006).
In addition to its interaction with Sbh1p or Sbh2p, some of the subunits of the OST also
interact with other components of the translocon. Stt3p, for example, was found to interact only
with Sec61p, whereas Ost4p interacts with all if the subunits of the translocon. Wbp1p interacts
strongly with Sec61p and Sbh1p, and weakly with Sss1p. An interaction with Sec61p and either Sbh1p
or Sss1p was also found for the Ost1p, Ost2p and Swp1p subunits, whereas Ost5p and Ost6p failed
to interact with Sbh1p or Sss1p (Chavan et al., 2005). Besides those multiple interactions between
the two complexes, it has also been demonstrated that the OST physically binds to the 60S
ribosomial subunit (Harada et al., 2009).
Each subunit of this complex has a specific function, and a model for the sequential steps of
the glycosylation has been proposed. Ost3p and Ost6p determine the association of a specific
isoform to a specific translocon, and Ost4p helps in the association of both complexes. Ost1p guides
the unfolded protein to the active site of the complex while Wbp1p presents the Dol-P-P-
oligosaccharide to the catalytic subunit of the OST. Stt3p scans the NC for glycosylatable sites and
catalyses the N-linked glycosylation of polypeptides, which reduces the affinity to the complex,
allowing the dissociation from this protein (Chavan and Lennarz, 2006).
3.2 - More post-translational events
Besides the N-Glycosylation of the nascent polypeptide chain, several modification and
events happen in the ER lumen, starting from the cleavage of the signal peptide that allows the
targeting to the ER by the SP on the lumenal side of the ER membrane (Alberts et al., 2002).
Among the modifications undergone by the protein after its translocation, there is the
formation of disulfide bonds. These bonds are essential for the stability of the tertiary and
quaternary structure of many proteins. The oxidation of the free sulfhydryl (SH-) groups of cysteines
(Cys) is catalyzed by PDI (Alberts et al., 2002; Lodish et al., 2000).
The addition of GPI-anchors to membrane proteins is another modification that the protein
has to go through. GPI-anchors are essential for the survival of S. cerevisiae for example, because
they are used to target certain mannoproteins for covalent incorporation into the β-glucan cell wall.
A precursor is pre-assembled in the ER membrane, before being attached to the newly synthesized

17
protein in the ER lumen and undergo lipid remodeling and/or carbohydrate side-chain modification
in the ER and the Golgi (Varki et al., 2009).
All those modifications, together with folding enzymes and different types of chaperone
molecules, help the folding of newly synthesized proteins, the prevention of inappropriate
interactions with other proteins (Braakman and Hebert, 2013).
3.3 - Protein inspection - ER Quality Control
Despite the process describe above, it can happen, that proteins are not able to reach their
native conformation (corresponding to the most energetically favorable state). These kind of
proteins, which are immature, misfolded or unfolded, are problematic because they are toxic for the
cell, as they cannot ensure their function correctly and can also aggregate (Ellgaard and Helenius,
2003). This misfolding can be caused by mutations, substoichiometric amounts of a binding partner
or a shortage of chaperone availability (Ruggiano et al., 2014). The role of the ERQC is to recognize
the misfolded proteins, and send them for further folding or degradation (Buchberger, 2014).
In order to recognize those proteins, the cell uses sensor molecules, such as the molecular
chaperone Kar2p, calnexin, calreticulin, glucose-regulated protein 94 (GRP94) and the thiol-
disulfide oxidoreductases PDI and ERp57 (Ellgaard and Helenius, 2003). BiP (Kar2p in yeast) is an
ER-resident chaperone molecule, which is a members of the Hsp70 family. It recognizes incorrectly
folded proteins by binding to exposed a.a. sequences that should be buried in the interior of
correctly folded polypeptide chains. In addition to its role in protein folding, it also plays a role in
the ERQC by preventing proteins from aggregating and retaining misfolded or unfolded proteins in
the ER (Alberts et al., 2002; Lodish et al., 2000)
The quality control (QC) is divided in a primary and a secondary QC. The primary QC works
at a general level, on all proteins, whereas the secondary QC is reserved for selected categories of
proteins, and is more specific. The general QC distinguishes between native and non-native proteins
basing on the exposure of hydrophobic regions, unpaired Cys residues and the tendency to
aggregate. It frequently happens that one or more of the sensor molecules bind to a protein with
even minor deviation from the native conformation (Ellgaard and Helenius, 2003).
A well-characterized primary QC is the calnexin/calreticulin cycle (Figure 4). This cycle
controls the quality of glycoproteins, by keeping uncorrectly folded ones in the ER until they reach
their native conformation, or targeting them to for degradation. Glucosidase I and II (Gls1/2p)
remove two glucose residues from the oligosaccharide, which leaves a monoglucosylated
glycoprotein. This protein binds to the globular lectin domain of celnexin or calreticulin. Both form
complexes with ERp57 and calreticulin-bound glycoproteins. Gls2p hydrolyses the glucose residue
from the monoglucosylated glycan, thus dissociating the latter from calnexin or clareticulin. The
correctly folded protein is then released from the ER to the Golgi in a transport vesicle, whereasl
misfolded proteins are reglucosylated by an UDP-glucose:glycoprotein glucosyltransferase (GT) to
enter the cycle again (Ellgaard and Helenius, 2003; Hammond and Helenius, 1995; Ware et al., 1995).
3.4 - The good, the bad and the misfolded - ER-Associated Degradation
It is estimated that 30% of the proteins in eukaryotic cells are misfolded during their
biosynthesis and degraded (Schubert et al., 2000). These misfolded proteins can form toxic
aggregates in the cell, and this is why cell have developed a way to eliminate them: the ERAD (Figure
5; Römisch, 2004).

18
Once they are recognized as un- or misfolded, these proteins are targeted for degradation in
the cytosol. In order to be retrotranslocated in the cytosol, proteins have to remain in a soluble
form, helped by Bip/Kar2p and other proteins (Römisch, 2005). This retrotranslocation is mediated
by Sec61p in association with other proteins and complexes (Wiertz et al., 1996).
Figure 4 - Calnexin/Calreticulin cycle. This is the specific cycle for folding and degradation of glycoproteins.
After the addition of an N-glycan to the polypeptide (1), two glucosidases, Gls1 and 2, remove two glucose
residues from the oligosaccharide (2). This monoglucosylated oligosaccharide then binds to the globular
lectin domain of calreticulin or calnexin (not shown), itself bound to ERp57 (3). The hydrolysis of the
remaining glucose residue by Gls2 allows the dissociation of the protein from the calreticulin/calnexin
complex (4), and if the protein is correctly folded, it is led to an ER exit site, for further processing in the
secretory pathway (5). In case the protein is still not correctly folded, an UDP glucose:glycoprotein GT adds a
new glucose residue to the glycan, so that it can enter the cycle again, until proper folding (6). Sometimes,
the protein fails to be correctly folded. In this case, an α1,2-mannosidase remove a mannose from the
oligossacharide chain (7), so that it can be targeted by the EDEM and retrotranslocated into the cytosol for
degradation (8). Adapted from Ellgaard and Helenius, 2003.

19
Proteins that have to be degraded need to be ubiquitinated. Ubiquitin is a 76 a.a. long
protein which is covalently attached to the target protein through the sequential action of activating
(E1), conjugating (E2) and ligase (E3) enzymes (Pickart, 2001; Vembar and Brodsky, 2008). This event
takes place in the cytosol, after the misfolded protein has been retrotranslocated (Römisch, 2005).
The best characterized ubiquitylation system is mediated by the E3 ligase complexes Hrd1p and
Doa10p (Hampton, 2002). Which complex is used for which protein is determined by the
localization of the misfolded protein, and in case of transmembrane proteins, the localization of
their misfolded domain: when it is localized in the cytoplasmic domain (ERAD-C substrates), the
degradation occurs via the Doa10 complex, whereas in the case of lumenal (ERAD-L substrates) or
intramembrane (ERAD-M substrates) misfolded domains, proteins are degradated via the Hrd1
complex (Ruggiano et al., 2014; Vembar and Brodsky, 2008). Ufd2p, Cdc48p, Rpn10p and Rpt5p
(subunits of the 19S RP of the proteasome), as well as Rad23p and Dsk2p mediate the
polyubiquitylation of the protein, and possibly mediate the transport to the proteasome proteolytic
core (Römisch, 2005).
The degradation of glycoproteins happens in a different way (Figure 4). A mannose in the middle
branch of the N-linked oligosaccharide is removed by the α1,2-mannosidase I, leading to an
association with ER degradation-enhancing 1,2-mannosidase-like protein (EDEM), localized on the
ER membrane (Ellgaard and Helenius, 2003; Vembar and Brodsky, 2008).
Proteins that are correctly folded can also be targeted by the ERAD. Their degradation is
highly regulated and can only happen in the presence of a specific signal. 3-hydroxy-3-
methylglutaryl acetyl-coenzyme-A reductase (HMGR) and squalene monooxygenase (SQLE or Erg1
in yeasts) are two enzymes of the sterol biosynthetic pathway, which suffer this type of degradation.
Indeed, this is part of a feedback inhibition system in order to prevent the accumulation of sterol
metabolites, which are toxic for the cell (Foresti et al., 2013).
3.5 - Stress in the ER and Unfolded Protein Response
The inactivation of ERAD results in an accumulation of misfolded proteins in the lumen or
membrane of the ER, inducing ER-stress. This ER-stress activates an intracellular signal
transduction pathway, called UPR (Walter and Ron, 2011), whose role is to maintain protein-folding
capacity in the ER (Korennykh and Walter, 2012).
The UPR involves one sensor in yeast, called Ire1, and three in higher eukaryotes, named
Ire1, PERK and ATF6 (Korennykh and Walter, 2012).
Ire1 is a kinase and consists of a N-terminal sensor domain in the ER lumen and a CDK2-like
Ser/Thr kinase domain fused to an unique C-terminal ribonuclease (RNase) domain in the cytosol.
When an unfolded/misfolded protein binds to its sensor domain, it activates the RNase domain,
that further cleaves HAC1 mRNA at two unconventional sites (Cox and Walter, 1996). The two
resulting exons are associated by tRNA ligase, and this induces the translation of functional Hac1
transcription factors. After entering the nucleus, the transcription factors activate the transcription
of UPR target genes, thus increasing the ER protein-folding capacity to the cell (Korennykh and
Walter, 2012).
Prolonged activity of the UPR indicates that ER-stress cannot be moderated, and induces
apoptosis (Tabas and Ron, 2011).

20
Figure 5 - ERAD Pathway. The first step in the ERAD is the recognition by sensor molecules of misfolded
proteins (1). Those misfolded proteins are then targeted to ligase enzymes (2), depending on the location of
the misfolded domain (Doa10 complex for cytosolic, Hrd1 for lumenal and intramembrane misfolded
proteins), in order to be retrotranslocated in the cytosol (3). Once in the cytosol, these proteins are oligo-
ubiquitylated by those ubiquitin ligase complexes (4), polyubiquitylated by a protein complex (5), and may
then be further led by Rad23p and Dsk2p to the 26S proteasome (6) for degradation (7). Modified from
Meusser et al., 2005.
4. AIM OF THIS STUDY
As it was described in the work of Soromani et al. (2012), T5 and S3 are two important
phosphorylation sites discovered on the cytosolic domain of Sbh1p. The mutation of Thr to Ala at
position 5 results in the destabilization of Sbh1p, but not in a hypophosphorylated protein. This
result suggests that the kinase responsible for the phosphorylation of the T5 was able to use other
residues as substrate, and the Ser at position 3 was proposed as a candidate for phosphorylation by
this kinase. The T5 phosphorylation site on Sbh1p evolved twice independently (in S. cerevisiae and
mammals) and is thus suggested to play a particularly important role, considering also that it is only
phosphorylated when part of the Sec61 complex.

21
Another domain of Sbh1p has been shown to have an important role: the TMD (Feng et al.,
2007). In fact, the expression of the TMD alone affects neither the functionality of the protein nor
its interaction with the Sec61 complex. The import of N-glycosylated α-factor precursor (3gpαf) by
the Sec61 complex was investigated in order to detect the defects in the functionality of Shb1p due
to the lack of its cytosolic or transmembrane domains. This led to the discovery that Δsbh1Δsbh2
cells show a reduced level of Msn1p due to constitutive translocation defects, further leading to a
reduced efficiency in the N-glycan trimming by glucosidase and mannosidase.
Following this finding that the TMD has an essential role, Zhao and Jäntti (2009) decided to
generate random mutations in the latter, in order to characterize the function of this domain. This
random mutagenesis led to the discovery that the TMD can be inactivated by the combination of
two point mutations: P54S and V57G.
This study is based on these findings, and it is aimed at the investigation of the influence of
these mutations in the phosphorylation sites and mutations in the TMD on the import of Gls1p, one
of the two other proteins that trims N-glycan for the ERQC.
As Sec61 is part of a bigger complex that allows the retrotranslocation of proteins into the
cytosol, these mutations might have affected the proper function of the ERAD, thus a part of this
work was also aimed at the study of possible defects caused by these mutations in the very important
process that is the ERAD.

22
II. Materials and Methods
1. MATERIALS
1.1 - Laboratory equipment, chemicals, reagents and consumables
Table 1 - Chemicals and reagents used in this study
Supplier Products
AGFA Healthcare GmbH
G153 Developer A/B
G354 Rapid Fixer
AnalaR® NORMAPUR® (part of VWR®
International GmbH)
Sodium Chloride
Glycine
Acetic Acid 100%
Hydrochloridric Acid
AppliChem GmbH
D(+) Glucose anhydrous
Sodium Dodecyl Sulfate
Cycloheximide
Tween® 20
Ampicillin Sodium Salt
Sodium Dihydrogen Phosphate
Becton, Dickinson and Company
Yeast Nitrogen Base without amino-acids
Yeast Nitrogen Base without amino-acids and
Ammonium Sulfate
Bacto™ Casamino-acids
Bio-Rad Laboratories, Inc. 30% Acrylamide/Bis solution, 37.5:1
CalbioChem® (part of Merck Millipore) Digitonin
Carl Roth® GmbH and Co. KG
Sodium Azide
Ammonium Persulfate

23
Sorbitol
Phenylmethylsulfonyl Fluoride
Yeast Extract
Peptone
Agar, Agar Kobe I
Triton X-100
Polyethyleneglycol 4000
Fisher Scientific (part of Thermo Fisher
Scientific)
Methanol
Formedium™
Complete Amino-acid Drop-out : -Leu (Kaiser
Mixture)
Complete Amino-acid Drop-out : -Leu/-Ura
(Kaiser Mixture)
Complete Amino-acid Drop-out : -Ura (Kaiser
Mixture)
GE Healthcare Protein-A Sepharose™ CL-4B
Grüssing GmbH Glycerol
Life Technologies (Part of Thermo Scientific)
Novex® MES Buffer NuPage
NuPage® 4-12 % Bis-Tris Gel 1.00 mm x 10 wells
(Novex®)
Novex® MOPS Buffer NuPage
Merck Millipore Magnesium Chloride
Perkin Elmer® Inc.
EXPRE35
S35
S Protein Labeling Mix, [35
S]-, 50
mM Tricine (pH 7,4), 10 mM 2-
Mercaptoethanol
[Methyl-14
C] Methylated Protein Molecular
Weight Marker
Roche GmbH
Complete ULTRA Tablets, Mini, EDTA-free,
EASYpack

24
Sigma-Aldrich®
Trizma Base®
Dimethylsulfoxyde
Lithium Acetate
Deoxyribonucleic Acid Sodium Salt from
Salmon Testes
Trypton
Ethanol
Tetramethylethylenediamine
Bromophenol Blue sodium salt for molecular
biology, for electrophoresis
Ethylenediaminetetraacetic Acid
Urea
Dithiothreitol
Sucofin® Skimmed Milk Powder
Thermo Scientific
Detection Reagent 1 Peroxide Solution
Detection Reagent 2 Luminol Enhancer
Solution
Restore™ Western Blot Stripping Buffer
Super Signal® West Dura Stable Peroxide
Buffer
Super Signal® West Dura Luminol/Enhancer
Solution
PageRuler Prestained Protein Ladder
Zentrales Chemikalien Lager Disodium Hydrogen Phosphate

25
Table 2 - Laboratory equipment used in this study
Company Product
Amersham Pharmacia Biotech Hypercassette™
Berthold technologies GmbH & Co. KG LB124 Contamination Monitor
Bio-Rad Laboratories, Inc.
PowerPac™ HC Power Supply
PowerPac™ 1000 Power Supply
PowerPac™ 3000 Power Supply
ChemiDoc™ XRS
Trans-Blot® Electrophoretic Transfer Cell
Model 583 Gel Dryer
Bio Spec Products Inc. Mini-Beadbeater-24
CAWO Solutions CAWOmat 2000 IR
Denver Instruments GmbH Ultra-basic 10 pH/mV Meter
Eppendorf AG
Minispin® Centrifuge
Thermomixer® Compact
Thermomixer® 5436
Microcentrifuge 5415R
Multipette® Plus
GE Healthcare
Image Eraser
Amersham Imager 600 RGB
Typhoon™ Trio Variable Mode Imager
Storage Phosphor Screens
Exposure Cassettes
Ultrospec 2100 pro UV/Visible
Spectrophotometer
Gilson N10478B, N23112C, EA56493, GC54948

26
Heraeus Instruments GmbH Hot Chamber
Hirschmann® Laborgeräte Pipetus® A41585G
IKA®-Werke GmbH & Co. KG RTC Basic (Laboratory overhead stirrer)
Infors HT Multitron Standard Incubation Shaker
Invitrogen™ (Life Technologies part of
ThermoFisher Scientific Inc.)
X-Cell Surelock™ Mini-Cell (Novex®)
Life Technologies Mini Gel Tank
LTF Labortechnik GmbH and Co. KG Unigeldryer 3545D
Merck Millipore Milli-Q® Integral Water Purification System
neoLab Migge Laborbedarf
Rotary wheel
ST5 (shaker)
New Brunswick Scientific (part of Eppendorf
AG)
Innova 4230 refrigerated incubator shaker
Peqlab NanoDrop 2000c Spectrophotometer
Rigal Bennett Luckham R100 Rotatest shaker
Sartorius AG
Balance
Precision Balance
Scientific Industries Inc. Vortex-Genie® 2
Scotsman® Ice Systems AF80 Flake Ice Machine
Sigma-Aldrich® SIGMA 2-16P Refrigerated Centrifuge
Stuart® (part of Bibby Scientific Limited) Mini orbital shaker SSM1
Systec GmbH
DX-150 Autoclave
VB-55 Autoclave
Vacuubrand GmbH+Co
Chemistry Vacuum System MZ 2C NT +2AK
VARIO®-SP Diaphragm Pump MZ 2 VARIO-SP
Zeiss West Germany Binocular Light Microscope

27
Table 3 - Consumables used in this study
Company Product
B. Braun Melsungen AG
Surgical Disposable Scalpel
Sterican® hypodermic needle Gr1/Gr16
Becton, Dickinson and Company
Plastipak™ Sterile Syringe Luer-Lock™
20 mL
Bio-Rad Laboratories, Inc.
Nitrocellulose Membrane (0,2 µM, 0,45 µM
pore size)
Biozym Scientific GmbH Surphob® Tips 1250 µL and 200 µL
Aluminium foil
Cover Glass 22 mm x 22 mm
Microscope Slides
Disposal bags
Costar® Stripette® 25 mL Serological Pipettes
Duran® Group
Graduated Cylinders
Erlenmeyer flask
Beaker
Solution Bottles
Eppendorf AG 2,5 mL, 5 mL, 12,5 mL Combitips
Fujifilm Fuji Medical X-Ray Film (Super RX)
GE Healthcare Whatman™ Paper
Greiner Bio-one International GmbH
Cellstar ® 10 mL serological pipettes
Cellstar® 14 mL and 50 mL falcons
Ultratips 1250 µL and 200 µL
Henke Sass Wolf
60 mL Norm-Ject® Luer-Lock™ Sterile
5 mL Soft-Ject® Luer Sterile

28
Life Technologies 1.0 mm/1.5 mm Gel Cassettes (Novex®)
Nalgene™ Labware (part of Thermo Scientific)
250 mL and 500 mL Rapid-Flow™ Filters
0,2 µm aPES membrane
neoLab Migge Laborbedarf
Blotting Paper Sheets 200 x 200 mm
330 g/m2
Pechiney Plastic Packaging Parafilm “M”®
Sarstedt
10 µL Inoculation Loops
5 mL Serological Pipettes
Petri dishes
Micro-tubes 1,5 mL, 2,0 mL
Plastic Cuvettes
Sartorius AG
Minisart® Sterile Syringe Filters (0.2µm pore
size)
Sigma-Aldrich
5 ml, 10 mL, 25 mL Volac® Graduated Glass
Pipettes
Glass beads, acid washed
VWR®
Latex gloves
500 mL Bottles and Filter Upper Cup
0,2 µm PES membrane sterile
Sterile syringe Filter 0,2 µm Cellulose Acetate
1.2 - Media and Buffers
Table 4 - Composition of the media used in this study
Name Composition
LB Medium (+Agar)
1% Trypton from Casein
0,5% Yeast Extract
0,5% Sodium Chloride (NaCl)

29
(1,5% Agar)
100 mg/L Ampicillin
YPD Medium (+Agar)
2 % Peptone from Casein
1 % Yeast Extract
(2 % Agar)
2% Glucose
Drop-Out Medium:
-LEU (+Agar)
(2 % Agar)
1,622 g/L Complete Amino-acid Drop-out: -Leu
6,7 g/L Yeast Nitrogen Base (YNB) without
amino-acids (a.a)
2 % Glucose
Drop-Out Medium:
-LEU/-URA (+Agar)
(2 % Agar)
1,546 g/L Complete Amino-acid Drop-out: -
Leu/-Ura
6,7 g/L YNB without (w/o) a.a
2 % Glucose
Drop-Out Medium:
-URA (+Agar)
(2 % Agar)
1,926 g/L Complete Amino-acid Drop-out: -
Ura
6,7 g/L YNB w/o a.a.
2 % Glucose
Minimal Medium
0,2% Casamino-Acids (CAA)
5% Glucose
Auxotrophic Amino-acids (ref. Annex 1)
6.7 g/L YNB without a.a
Table 5 - Composition of the solutions for yeast transformation
Name Composition
10x LiAc pH 7,5
1 M Lithium Acetate (LiAc)
Acetic Acid to optimize pH to 7,5

30
Table 6 - Composition of the solutions for cell extract preparation
Name Composition
DTT 1 M Dithiothreitol (DTT)
2x Sample Buffer
50 mM Tris-HCl pH 7,5
5 % Sodium Dodecyl Sulfate (SDS)
20 % Glycerol
2 % Bromophenol Blue
Table 7 - Composition of the solutions for SDS-PAGE and Western Blot
Name Composition
10x Tris-Glycine
25 mM Trizma Base
2 M Glycine
Transfer Buffer
1x Tris-Glycine
2 % Methanol (MetOH)
10x TE pH 7,5
10 mM Ethylenediaminetetraacetic Acid
(EDTA)
Hydrochloridric Acid (HCl) to optimize pH to
7,5
1M Tris-HCl pH 7,5
121,14 g/L Trizma Base
HCl to optimize pH to 7,5
50% PEG4000-solution 500 g/L Polyethyleneglycol (PEG) 4000
LiAc/TE-solution
1x LiAc pH 7,5
1x TE pH 7,5
PEG solution
1x LiAc pH 7,5
1x TE pH 7,5
40% PEG4000-solution

31
1 % SDS
10x Tris-Buffered Saline (TBS) pH 7,4
0,5 M Tris HCl
1,5 M NaCl
HCl to optimize pH to 7,4
TBS-T
1x TBS pH7,4
1 % Tween-20
Milk Buffer
1x TBS pH 7,4
5 % non-fat dry milk
1 % Tween 20
5 mM Sodium Azide (NaN3)
10x SDS-Running Buffer
250 mM Tris-HCl
2 M Glycine
1 % SDS
SDS-PAGE Gel 7,5%
7.5% Resolving Gel
5% Stacking gel
4,7925 mL Water (H2O)
2,5 mL 30% Acrylamide
2,5 mL 1,5 M Tris-HCl pH 8,8
0,1 mL 10% SDS
0,1 mL 10% Ammonium Persulfate (APS)
7,5 µL Tetramethylethylenediamine (TEMED
2,1 mL H2O
0,5 mL 30% Acrylamide
0,38 mL 1 M Tris-HCl pH 6,8
30 μL 10% SDS
30 μL 10% APS
3 μL TEMED
Table 8 - Composition of the solutions for pulse experiments
Name Composition
Labelling Medium
5 % Glucose
Autotrophic a.a

32
6,7 g/L YNB without Ammonium Sulfate and
a.a
Tris-Azide
20 mm NaN3
Resuspension Buffer
10 mM DTT
20 mM NaN3
Lysis Buffer
2 % SDS
1 mM DTT
1 mM Phenylmethanesulfonyl Fluoride (PMSF)
IP Buffer
150 mM NaCl
1 % Triton X-100 (TX-100)
0,1 % SDS
1mM PMSF
2 mM NaN3
Urea Wash
2 M Urea
200 mM NaCl
1 % TX-100
2 mM NaN3
ConA Wash
500 mM NaCl
1 % TX-100
2 mM NaN3
Tris-NaCl Wash
50 mM NaCl
2 mM NaN3
Fixing Solution 1 10 % Acetic Acid

33
40 % MetOH
2 % Glycerol
Fixing Solution 2
50 % MetOH
1 % Glycerol
Table 9 - Composition of the solutions for native immunoprecipitation
Name Composition
Lysis Buffer
200 mM Sorbitol
100 mM NaCl
25 mM Sodium Phosphate Buffer pH 7,4
1 mM Magnesium Chloride (MgCl2)
1 mM PMSF
1x Protease Inhibitor Cocktail
10% Glycerol
Sodium Phosphate Buffer pH 7,4
80% 1 M Disodium Hydrogen Phosphate
(Na2HPO4)
20% 1 M Sodium Dihydrogen Phosphate
(NaH2PO4)
1.3 - Bacterial and yeast strains
Table 10 - Escherichia coli (E.coli) strains used in this study
Strain Genotype Source
DH5α
F endA1 glnV44 thi-1 recA1 relA1
gyrA96 deoR nupG Φ80dlacZΔM15
Δ(lacZYA-argF)U169, hsdR17(rK
-
mK
+
),
λ–
Hanahan, 1983
XL-1 Blue
recA1 endA1 gyrA96 thi-1 hsdR17
supE44 relA1 lac [F´ proAB
lacIqZΔM15 Tn10 (Tetr)]
Stratagene: cat.#200249
KRB319 pDN431 (CPY*HA; URA3) Ng et al., 2000

34
Table 11 - S. cerevisiae strains used in this study
Strain Genotype Source
KRY293
leu2-3,112 ura3-52 seb::URA3,
MATα
J.H. Toikkanen
KRY294
leu2-3,112 ura3-52 seb2::G418R,
MATa
J.H. Toikkanen
KRY585 leu2-3, 113 ura3-52, MATa P. Novick, J. Jäntti
KRY588
seb1::KanMx seb2::hphMx leu2-
3,112 ura3-52 GAL+, MATa
D. Feng, J. Jäntti
KRY879
ade2-1 ura3-1 his3-11,15 leu2-
3,112 trp1-1 can1-100
der1::natNT2
C. Servas (Römisch Lab)
KRB320 pDN436 (CPY*HA; LEU2) Ng et al., 2000
KRB536 pRS415 (in XL-1 Blue) Sikorski et al., 1987
KRB689 SEB1pRS415 (in DH5α) C. Soromani
KRB746 S3ApRS415 (in DH5α) C. Soromani
KRB747 T5ApRS415 (in DH5α) C. Soromani
KRB987 YEpSBH1TM(P54S,V57G) (in DH5α) Zhao et al., 2007
KRB1032 pRS415 SBH1 S3A/T5A (in XL-1 Blue) J. Simon
KRB1046
pRS415 SBH1 S3A TMDM (in XL-1
Blue)
J. Simon
KRB1047
pRS415 SBH1 T5A TMDM (in XL-1
Blue)
J. Simon
KRB1048
pRS415 SBH1 S3A/T5A TMDM (in
XL-1 Blue)
J. Simon
KRB1050 pRS415 SBH1 TMDM (in XL-1 Blue) J. Simon

35
KRY1015
[pRS415 SBH1 S3A/T5A]
J. Simon
KRY1019
[pRS415 SBH1 S3A TMDM]
J. Simon
KRY1020
[pRS415 SBH1 T5A TMDM]
J. Simon
KRY1021
[pRS415 SBH1 S3A/T5A
TMDM]
J. Simon
KRY1049
[pRS415 SBH1 TMDM]
J. Simon
1.4 - Plasmids
Table 12 - Plasmids used in this study
Plasmid Marker / Selection Carrier Strain Reference/Source
pRS415 LEU2 / Amp KRB536 Sikorski et al. (1987)
pDN431-CPY*HA URA3 / Amp KRB319 Ng, et al. (2000)
pDN436-CPY*HA LEU2 / Amp KRB320 Ng, et al. (2000)
S3ApRS415 LEU2 / Amp KRB746 C. Soromani
SEB1pRS415 LEU2 / Amp KRB689 C. Soromani
T5ApRS415 LEU2 / Amp KRB747 C. Soromani
YEpSBH1TM (P54S,V57G) LEU2 / Amp KRB987 Zhao et al. (2007)
pRS415 SBH1 S3A/T5A LEU2 / Amp KRB1032 J. Simon
pRS415 SBH1 S3A TMDM LEU2 / Amp KRB1046 J. Simon
pRS415 SBH1 T5A TMDM LEU2 / Amp KRB1047 J. Simon

36
1.5 - Antibodies
Table 13 - Antibodies used in this study
Antibody Protein detected
Molecular
Weight of the
Protein
Use and
Working
Dilution
Source
anti-Cdc48p
(rabbit)
Cell Division
Cycle
92 kDa WB – 1:1250 N. Zheng
anti-CPYp
(rabbit)
Carboxypeptidase
Y
pCPY = 59 kDa;
p1CPY = 67 kDa;
p2CPY = 69 kDa;
mCPY = 61 kDa
WB - 1:3000 KB Römisch
anti-Gls1p
(rabbit)
Glucosidase 1 96 kDa
WB – 1:2000
IP – 1:100
A. Shibuya
anti-Rpn12p
(rabbit)
Proteasome
Regulatory
Particle Non-
ATPase
35 kDa WB – 1:2000 K. Kalies
anti-Sbh1p
(rabbit)
Sec61 beta
homolog 1 (N-
terminus)
9 kDa
(12 kDa on Gel)
WB – 1:2500 KB Römisch
anti-Sbh1p
(rabbit)
Sec61 beta
homolog 1 (a.a.
10-23)
9 kDa
(12 kDa on Gel)
WB – 1:2500
IP – 1:200
Pineda Antikörper
Service
anti-Sec61p
(rabbit)
Sec61 (N-
terminus)
52 kDa
(42 kDa on Gel)
WB – 1:2500 KB Römisch
anti-Wbp1p
(rabbit)
Wheat germ
agglutinin-
Binding Protein
49 kDa
WB – 1:2000
IP – 1:200
M. Aebi
pRS415 SBH1 S3A/T5A
TMDM
LEU2 / Amp KRB1048 J. Simon
pRS415 SBH1 TMDM LEU2 / Amp KRB1050 J. Simon

37
anti-rabbit (HRP
conjugated)
(goat)
Rabbit IgG - WB – 1:10000
Rockland™: cat. #
611-1302
1.6 - Softwares
Table 14 - Softwares used in this study
Company Software
Bio-Rad Laboratories, Inc. Quantity One 4.6.2 Basic
National Institute of Health ImageJ
GE Healthcare
Image Quant™ TL 8.1
Typhoon Scanner Control
2. METHODS
2.1 - Sterilization
All glassware and media were sterilized by autoclaving at 100 kPa and 120°C for 20 min if not
stated otherwise.
2.2 - Growth of S. cerevisiae
S. cerevisiae cells were grown at 30°C in YPD with continuous shaking at 200 rpm or on YPD
or drop-out plates at 30°C if not stated otherwise (Table 4)
2.3 - Growth of E. coli
E. coli cells were grown at 37°C, if not stated otherwise, in LB medium with continuous
shaking at 220 rpm or on LB medium plates (Table 4).
2.4 - Plasmid Extraction
The kit Invisorb® Spin Plasmid Mini Two (Stratec Molecular, Birkenfeld, Germany) was used
for plasmid extraction from bacteria, to later transform yeast cells with it.
To extract the needed plasmid, 2 mL of an E. coli overnight (o.N.) culture were transferred
in a micro-tube and centrifuged for 1 min at 13.000 rpm to pellet the cells. After removing the
supernatant, the cells were resuspended in 250 µL of Solution A, to weaken the cell walls and then
250 µL of Solution B were added to perform the lysis. Finally, 250 µL of Solution C were added to
stop the lysis, and the samples were centrifuged for 5 min at 13.000 rpm. The supernatant was then
transferred onto a Spin Filter, incubated for 1 min at room temperature (RT) and centrifuged for 1
min at 13.000 rpm. After the filtrate was discarded, 750 µL of Wash Solution were added and the
samples were centrifuged at 13.000 rpm for 1 min, and then centrifuged again at 13.000 rpm for 3

38
min, to remove the residual ethanol (EtOH) from the filter. Finally, 50 µL of hot (~ 65°C) sterile
water was added in the middle of the filter, incubated 1 min at RT and centrifuged at 13.000 rpm for
1 min to elute the plasmid.
Plasmid concentration was determined using the NanoDrop 200oc spectrophotometer from
Manfred Schmitt’s laboratory.
2.5 - Transformation of S. cerevisiae
Yeast cells were transformed in order to study the influence of given mutations on specific
proteins or organelles.
The protocol used for yeast transformation was the Lithium Acetate (LiAc) Transformation
protocol. In order to perform the transformation, 2 mL of a S. cerevisiae o.N. culture were harvested
at 3.000 rpm for 2 min. The pellet was then washed with 1 mL of LiAc/TE Buffer and centrifuged for
2 min at 3.000 rpm. The pellet was then resuspended in 100 µL of LiAc/TE and 20 µL of denatured
Carrier-DNA (salmon sperm), 1 µg of plasmid DNA (pDNA), 600 µL of PEG-Buffer and 50 µL of 10x
LiAc were added. The samples were incubated for 1 h at 30°C. After this incubation, 20 µL of DMSO
were added, and the samples were incubated for 15 min at 42°C. They were then centrifuged for 2
min at 3.000 rpm and washed two times with 1 mL of 1x TE. Finally the pellet was resuspended in
100 µL of 1x TE and plated on corresponding auxotrophy plates.
2.6 - Preparation of Cell Extracts
Cell extracts were prepared in order to investigate soluble or membrane proteins of the
strains employed in this study.
The preparation of whole-cell extracts requires an OD600 between 1 and 2 (when yeast cells
are in the exponential growth phase).
The cells were concentrated to 2 OD600/mL. 1 mL was taken and centrifuged at 13.000 rpm
for 1 min. They were then resuspended in 1 mL of sterile water and centrifuged at 13.000 rpm for 1
min. The pellet was then resuspended in 100 µL of 200 mM DTT 2x Sample Buffer. Half of the volume
of Glass beads was added and the lysis was performed with a Beadbeater (Bio Spec Products Inc.,
Bartlesville, USA). Two 1 min cycles of disruption were performed with 1 min of incubation at 4°C
between each one. The samples were finally denatured for 5 min at 95°C or for 10 min at 65°C if
soluble or membrane proteins had to be investigated, respectively.
2.7 - Protein Gel Electrophoresis and Western Blot Analysis
2.7.1 - Protein Gel Electrophoresis
Protein Gel Electrophoresis was employed to resolve proteins according to their molecular
weight.
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was carried using NuPage® 4-12 % Bis-
Tris precast gels or self-made 7,5 % SDS-Gels. 6 µL Page Ruler Prestained Protein Ladder (Thermo
Scientific, Waltham, USA) and 20 µL of each sample were loaded. Precast gels were ran in 1x MOPS
buffer (for better resolution of high molecular weight proteins) or 1x MES buffer (for better
resolution of low molecular weight proteins), self-made SDS-gels were ran in 1x Running Buffer, at
80 V for 15 min and then at 160 V for 1 h.

39
2.7.2 - Western Blot Analysis
Western Blotting was employed to identify the desired proteins using appropriate
antibodies.
Proteins were transferred onto nitrocellulose membranes. The blot was assembled using the
nitrocellulose membrane as well as Whatman® Paper and sponges soaked in Transfer Buffer in a
Transfer Cell (Bio-Rad Laboratories, München, Germany) at 4°C for 1 h 30 min at 100 V, or o.N. at
0.3 A.
Following the transfer, the membranes were blocked in Milk Buffer under shaking for 1 h,
at RT or o.N. at 4°C. The membranes were then incubated under shaking with the specific antibody
(diluted in Milk Buffer), for 2 h at RT or at 4°C o.N.. The membranes were subsequently washed
twice in Milk Buffer and twice in TBS-T for 10 min. They were then incubated with the secondary
antibody (anti-rabbit, diluted in TBS-T) for 1 h under shaking at RT, and later washed 5 or 6 times
for 5 min with TBST. Finally, the membranes were covered with Detection Reagent 1 Peroxide
Solution and Detection Reagent 2 Luminol Enhancer Solution or Super Signal® West Dura Stable
Peroxide Buffer and Super Signal® West Dura Luminol/Enhancer Solution (Thermo Scientific,
Waltham, USA) and revealed by enhanced chemoluminescence (ECL) in the CAWOmat 2000
(CAWO Solutions, Schrobenhausen, Germany), ChemiDoc XRS (Bio-Rad Laboratories, München,
Germany) or Amersham Imager 600 RGB (GE Healthcare, Freiburg, Germany).
2.7.3 - Membrane stripping
In order to remove primary and secondary antibodies from a WB membrane and reprobe it
with a different primary antibody, the membranes were washed in TBS-T for 5 to 10 min, and then
incubated in Restore™ Western Blot Stripping Buffer (Thermo Scientific, Waltham, USA) for 15 min
at RT. Afterwards, they were washed two times 5 min with TBS-T and the immunoblotting was
performed again.
2.8 - Cycloheximide Chase Experiments
Cycloheximide chase was performed to investigate ERAD defects, using a misfolded variant
of the protein CPY.
Strains of interest were grown o.N. at 30°C to an OD600 of 1-2. 0,2 mg/mL cycloheximide was
added to the liquid cultures. At t=0, 1 mL duplicates, with a concentration of 2 OD600/mL were taken.
Regular amount of cells were removed every 15 min for 60 min.
Cell extracts were then prepared as described in 2.6.
2.9 - Pulse Experiments
2.9.1 - Pulse Labeling
The Pulse labeling experiment was employed to identify desired proteins with more
specificity than the Western Blot, using radioactive peptides and immunoprecipitation. The labeling
mix is composed of 35
S-labeled L-methionine (Met) and 35
S L-cysteine (Cys). Since the cells are taken
while they are quite actively proliferating, because they are in the beginning of the exponential
phase, these labeled amino-acids will be assimilated and incorporated during protein synthesis. This
causes all new synthesized proteins to be 35
S labelled, which enables a very sensible detection of
newly synthesized proteins.

40
Strains of interest were grown o.N. in Minimal Medium at 30°C, to an OD600 of 0,5-1,5. The
cells were harvested at 4.500 g for 5 min and washed twice with 10 mL Labeling Medium. They were
then concentrated to 6 OD600/mL. 250 µL were taken in duplicate from each culture, in order to
have an OD600 of 1,5 in each sample. They were then incubated at 30°C for 20 min under shaking
(800 rpm). 4,5 µL of Labeling Mix (EXPRE35
S35
S Protein Labeling Mix, Perkin Elmer) were added to
each sample. The labeling was stopped after 5 min using 750 μL of ice-cold Tris-Azide. The samples
were then centrifuged for 1 min at 13.000 rpm, and washed again with 1 mL of Tris-Azide. The pellets
were resuspended in 1 mL of Resuspension Buffer and incubated at RT for 10min. After a 13.000 rpm
1 min centrifugation, 150 μL of Lysis Buffer and half of the volume of Glass beads were added and
the cells were lysed using a Beadbeater. Three 1 min cycles of disruption were performed with 1 min
of incubation at RT between each one. Samples were then centrifuged (1 min, 13.000 rpm) and
incubated at 95°C for 5 minutes. Finally, the samples were washed three times with 250 µL of IP
Buffer w/o SDS and the supernatants were collected.
2.9.2 - Immunoprecipitation
Supernatants collected in the previous step were incubated on a rotary wheel for 30 min at
RT with 60 μL of Protein-A Sepharose for a pre-clearing. They were then centrifuged (1 min, 13.000
rpm) and the supernatants were collected. 5 µL to 10 µL of specific antibody were added, as well as
60 μL of Protein-A Sepharose and 1 mL of IP Buffer with SDS. The samples were incubated for 2 h
at RT (or overnight at 4°C) on a rotary wheel. After a quick spin (1 min, 13.000 rpm), the supernatants
were collected in a new tube and frozen for later immunoprecipitation with different antibodies.
Each pellet was washed two times with 1 mL of IP Buffer, once with 1 mL of Urea Wash, 1 mL of
ConA Wash, 1 mL of Tris-NaCl. 20 μL of 200 mM DTT 2x Sample Buffer were then added and the
samples were heated for 5 min at 95°C.
2.9.3 - Gel Electrophoresis and Revealing
The samples were resolved as described in 2.7.1.
25 µL of each sample and 6-8 µL Page Ruler Prestained Protein Ladder/[Methyl-14
C]
Methylated Protein Molecular Weight Marker (Perkin Elmer) were loaded
Afterwards, the gel was fixed in Fixing Solution 1 for 15 min and in Fixing Solution 2
for 30 min.
Finally, the gel was dried for 2 h at 80°C in gel dryer (583 Gel Dryer, Bio-Rad Laboratories,
München, Germany) and a phosphor plate (GE Healthcare, Freiburg, Germany) was exposed to it
for 12-24 h.
2.10 - Native Immunoprecipitation
Native immunoprecipitation experiment was carried to study the interaction between Sbh1p
and the OST.
Strains of interest were grown o.N. at 30°C, to an OD600 of 1-2. Cells were concentrated to 2
OD600/mL, and 5 samples of 1 mL each were taken. Samples were centrifuged at 8.000 rpm for 2 min.
They were then resuspended in 50 μL of 100 mM Tris-HCl pH 9,4, incubated for 10 min at RT and
centrifuged at 8.000 rpm for 2 min.
All the subsequent steps were performed on ice or at 4°C.

41
The samples were resuspended in 100 μL of Lysis Buffer w/o Glycerol, and half of the volume
of Glass Beads was added. The cells were lysed using a Beadbeater. Three 1 min cycles of disruption
were performed with 1 min of incubation at RT between each one. After a short centrifugation (2.000
rpm for 1 min), all supernatants of the same strain were taken together in a new tube, and
centrifuged for 10 min at 13.000 rpm. The pellet was resuspended in 50 µL of Lysis Buffer. 200 µL of
Lysis Buffer with 3,75% digitonin were then added, and the samples were incubated on ice for 30
min. After another centrifugation at 13.000 for 10 min, the supernatant was taken in a new tube and
brought to 1 mL with Lysis Buffer. The Protein-A Sepharose was washed five to six times in Lysis
Buffer to be equilibrated. Then, 60 μL of Protein-A Sepharose were added to the supernatants that
were then incubated on a rotary wheel for 30 min at 4°C for pre-clearing. The samples were then
centrifuged and the supernatants were taken in a new tube. 5 µL to 10 µL of specific antibody were
added, as well as 60 μL of Protein A Sepharose and 1 mL of Lysis Buffer, and incubated for 2-4 h at
4°C on a rotary wheel. After a quick spin (1 min, 13.000 rpm), the pellet was washed five to six times
with Lysis Buffer. Finally, 20 µL of 2x Sample Buffer were added, and the samples were heated for 10
min at 65°C.
The samples were then resolved using an SDS-Gel and the proteins were then transferred
onto nitrocellulose for immunoblotting (ref. 2.7.2).

42
III. Results
1. IMPORT OF GLUCOSIDASE I INTO THE ER
Glucosidase I is an important enzyme that plays a role in the proper folding of glycoproteins.
It removes the α-1,2-linked glucose residue from the oligosaccharide chain of glycoproteins, in order
to allow a second glucosidase (Gls2) to remove the second glucose residue from the chain. If Gls1p
doesn’t remove the first glucose residue, Gls2 won’t have access to the second glucose, the
glycoprotein will not be recognized by the ERQC, and be targeted to the ERAD for degradation by
the 26S proteasome (ref. I.3.3).
Thus it is important that Gls1 is correctly imported into the ER, and this is what we
investigated in the following mutants.
1.1 - Phosphorylation- and transmembrane double mutants
As it was described in the work of Soromani et al. (2012), T5 and S3 are two important
phosphorylation sites discovered on the cytosolic domain of Sbh1p. The mutation of Thr to Ala at
position 5 results in a destabilization of Sbh1p, but not in a hypophosphorylated protein. This result
suggested that the kinase responsible for the phosphorylation of the T5 was able to use other residue
as substrate, and the Ser at position 3 was proposed as a candidate. The T5 phosphorylation site on
Sbh1p evolved twice independently (in S. cerevisiae and mammals) and is thus suggested to play a
particularly important role, even more because it is only phosphorylated when part of the Sec61
complex.
Another domain of Sbh1p has been shown to have an important role: the TMD (Feng et al.,
2007). In fact, the expression of the TMD alone neither affect the functionality of the protein nor its
interaction with the Sec61 complex. The import of N-glycosylated α-factor precursor (3gpαf) by the
Sec61 complex was investigated in Δsbh1Δsbh2 cells in order to detect defects in the functionality of
Shb1p due to the lacking of its cytosolic and transmembrane domains. This led to the discovery that
Δsbh1Δsbh2 cells show a reduced level of Msn1p due to constitutive translocation defect, further
leading to a defect in the N-glycan trimming by glucosidase and mannosidase.
As the role of the TMD wasn’t defined, Zhao and Jäntti (2009) decided to characterize the
function of the latter. They thus generated random mutations in this domain, to observe how they
influenced the protein translocation. This led to the discovery, that a combination of two point
mutations in the TMD (P54S, V57G) results in the inactivation of the transmembrane domain.
The import of Gls1 in T5A, S3A, as well as in the mutant carrying two point mutations in the
transmembrane domain (P54S, V57G; Zhao and Jäntti, 2009) was investigated in Δsbh1Δsbh2 cells,
in order to identify a defect in the translocation of this enzyme.
On Figure 6, the results of the performed Western Blot are presented for the three mutants
mentioned above, as well as strains carrying the wild-type SBH1 gene (WT SBH1), the vector control
(pRS415) and the WT strain expressing both Sbh1p and Sbh2p.
The WT strain clearly shows an intense band, indicating that Gls1 was correctly imported.
The S3A mutant only displayed a mild defect in the import of Gls1, while a more pronounced defect
was shown by the T5A and the transmembrane double mutant (TmDM). The WT SBH1 and pRS415
strains manifest a very strong defect in the import of Gls1.

43
Figure 6 - Import of Gls1 in SBH1 mutant strains. Transformants and the corresponding WT were grown at
30°C to an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-PAGE. After
transfer on nitrocellulose membrane, they were immunoblotted with anti-Gls1p and anti-Cdc48p (loading
control) antibodies. The P54S, V57G strain was only expressing the TMD.
The following graph (Figure 7) represents the average amount of Gls1 in the cell for all strains
for three repetitions of the experiment.
Figure 7 - Amount of Gls1 in the cell for different SBH1 mutants. Quantification was performed using ImageJ.
Error bars indicate the standard error.
Less Gls1 is present in the mutants compared to the WT, but the strain carrying the S3A
mutation seems to have the stronger import of Gls1 even though the error bar indicate that it might
not be significant. The empty plasmid also shows a stronger import than the other mutants. The
T5A and WT SBH1 strains carry a slightly lower amount of Gls1, whereas the P54S, V57G mutant
clearly present a very reduced import of this protein.
In order to have a more precise idea of the behavior of these mutants, a Pulse experiment
has been performed. However, the immunoprecipitation was unsuccesful, and no signal could be
detected.

44
The TmDM used for all experiments only contains the TMD, in p425ADH. The TmDM used
for the experiment shown in Figure 8 was generated by Julien Simon in the same plasmid (pRS415)
as the other mutants, and expresses the whole Sbh1 protein containing the transmembrane
mutations.
As expected, the WT strain shows a robust import of Gls1 in the ER, as do the T5A strain and
the TmDM. The WT SBH1 strain also shows a strong band, but a slightly smaller amount of Gls1 was
imported. The S3A strain seems to import slightly less Gls1 than the WT SBH1, while a weak band
was displayed by the vector control.
Figure 8 - Import of Gls1 in SBH1 mutant strains. Transformants and the corresponding WT were grown at 30°C
to an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-PAGE. After
transfer on nitrocellulose membrane, they were immunoblotted with anti-Gls1p and anti-Cdc48p (loading
control) antibodies.
Thus, we observe a very different behavior between the TmDM expressing the whole
Sbh1 protein and the one expressing only the TMD. This mutant was generated at the end of Julien
Simon’s Bachelorthesis, I was thus unable to repeat this experiment due to time constraints.
1.2 - Combined mutants
The mutants used for the second part of my Bachelorthesis were generated by Julien Simon
using site-directed mutagenesis. These mutants correspond to combinations of the previously
tested mutations: S3A/T5A; TmDM + S3A; TmDM + T5A, TmDM + S3A/T5A (ref. II.1.3 - Tables
10/11/12).
The Western Blot Analysis of those mutants was performed in order to investigate the effect
of combined mutations on the import of Gls1 into the ER lumen.
The results of this experiments are shown in Figure 9. Since most of the mutations are
combined with the mutations in the TMD, the TmDM expressing the whole Sbh1 protein was used
as a control in addition to the controls used for the previous experiment.
The WT strain does not show any Gls1 import defect; the TmDM and S3A+TmDM are almost
comparable to the WT in the import of this protein. A slightly reduced import into the ER is
observed in the T5A+TmDM, S3A/T5A+TmDM and the WT SBH1 strains. The intensity of the band
in the S3A/T5A and pRS415 strains are very low, indicating a strong import defect of Gls1.
In order to have a better understanding of the results, the bands were normalized against
the corresponding loading control. The mean results for both duplicates of this experiment are
shown in Figure 10.

45
Figure 9 - Import of Gls1 in SBH1 combined mutant strains. Transformants and the corresponding WT were
grown at 30°C to an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-
PAGE. After transfer on nitrocellulose membrane, they were immunoblotted with anti-Gls1p and anti-Cdc48p
(loading control) antibodies.
Figure 10 - Amount of Gls1 in the cell for different combined SBH1 mutants. Quantification was performed using
ImageJ. Error bars indicate the standard error.
Less Gls1 is expressed in the mutants compared to the WT, but the strain carrying the
S3A/T5A+TmDM mutation seems to have a stronger import of Gls1 than the other mutants. The WT
SBH1 strain also seems to import more Gls1 than the other mutants, which is consistent with the
expression of the WT protein. The S3A+TmDM and T5A+TmDM strains show a similar import of
Gls1, and the amount imported by the TmDM is also comparable. The empty plasmid strain and the
S3A/T5A mutant, however, manifest a strong defect in the import of Gls1, compared to the other
mutants and to the WT.
The quantification of this experiment shows large error bars. In order to have a more precise
idea of the behavior of these mutants, the experiment has to be repeated. Julien Simon managed to
generate two of those mutants only in the penultimate week of our Bachelorthesis. Due to time
constraints, I was unable to perform more repetitions. For the same reason, a Pulse experiment of
these mutants could not be conducted.

46
2. OLIGOSACCHARYL TRANSFERASE COMPLEX AND SBH1P
Several forms of CPY can be found in the cell. There is a proCPY (pCPY) polypeptide of
59 kDa found in the cytosol, an ER form (p1CPY) of 67 kDa, a Golgi form (p2CPY) of 69 kDa, and
finally the mature form (mCPY) of 61 kDa. Their molecular weight changes when they are
unglycosylated: p2CPY is then 59 kDa, as well as p1CPY, and mCPY is 51 kDa (Stevens et al., 1982).
CPYp was used as a loading control for the immunoblotting of Gls1, but we noticed that for
two of the strains, several forms of CPY were present in the cell (Figure 11), so it couldn’t be further
used as loading control, as it seemed that the mutations affected its import. That is the reason why
we used Cdc48p as a loading control afterwards.
Figure 11 - Import of CPY in SBH1 mutants. Transformants and the corresponding WT were grown at 30°C to
an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-PAGE. After transfer
on nitrocellulose membrane, they were immunoblotted with anti-Gls1p and anti-Cdc48p (loading control)
antibodies. The P54S, V57G strain was expressing the whole SBH1 protein containing those mutations in the
TMD.
For every strain, at least two bands are detected, obviously corresponding to p2CPY (69 kDa)
and p1CPY (67 kDa). Nevertheless, there are two more bands detected for the TmDM and the pRS415
strain. They probably correspond to the mCPY (61 kDa), the pCPY (59 kDa) and the unglycosylated
forms of p1CPY and p2CPY (both 59 kDa).
This led us to think that the double mutation in the transmembrane domain of Sbh1p might
lower the interaction of the latter with the OST, thus resulting in an underglycosylation of this
protein. In order to test this hypothesis, a native IP of the Sec61 complex interacting with the OST
has been performed.
The native IP (ref. II.2.10) was first performed using antibodies directed against Wbp1p on
the TmDM and the WT strain. Anti-Wbp1p and anti-Sbh1p antibodies were then used for the
immunoblotting. Unfortunately, we couldn’t detect any Sbh1p in neither of the strains, and we were
only able to detect the heavy chain (HC) of the antibody (not shown).
This experiment was repeated using monospecific anti-Sbh1p antibodies for the IP instead
of anti-Wbp1p. The immunoblotting was performed using anti-Wbp1p and monospecific anti-Sbh1p
antibodies. The same results as in the first experiment were obtained: we could only detect the HC
of the antibody (not shown).

47
3. ER-ASSOCIATED DEGRADATION DEFECTS
When proteins are not able to reach their native conformation, they are degraded by the 26S
proteasome in the cytosol following the ERAD pathway. This prevents the accumulation of toxic
substrates in the ER, which can lead to cell death (ref. I.3.4).
Sometimes, this degradation pathway doesn’t work properly, and there is an accumulation
of misfolded proteins in the lumen of the ER. We investigated the ERAD of several mutant strains,
following the degradation of a mutant form of CPY, named CPY*.
3.1 - Phosphorylation- and transmembrane double mutants
In order to identify ERAD defects in the mutant strains we generated, cycloheximide chase
experiments were performed (ref. II.2.8). The cycloheximide blocks the translation of new mRNAs,
thus allowing us to follow the destiny of the already translated and translocated proteins. The
mutant variant of CPY (G225R; allele: prc1-1), CPY*, is recognized by the ERQC, retrotranslocated in
the cytosol and degraded by the 26S proteasome (Finger et al., 1993).
Strains containing the mutations of interest (S3A, T5A and the transmembrane double
mutation), as well as three controls (WT SBH1, pRS415 and Δder1) were first transformed with the
plasmid pDN431 carrying the gene encoding the mutated variant of CPY, tagged with Hemagglutinin
(HA).
The results of one of these experiments are shown in Figure 12. It has been previously
demonstrated that a mutation in DER1 results in an ERAD defect, thus the Δder1 mutant was used
as a positive control (Knop et al., 1996).
After 15 min of chase, half of the CPY* carried by the WT SBH1 strain (red line) was already
degraded; in the S3A (purple line) and T5A (light blue line) mutants the half-life of CPY* was
extended to 20 min. Nevertheless, about 90% of the CPY* in those cells was degraded after 45 min,
and 95 to 97 % after 60 min. These data indicate that those mutants do not show any ERAD defect.
After only 15 min, half of the CPY* was degraded in the Δder1 (orange line) cells but
afterwards it wasn’t degraded anymore as the amount stays relatively identical. After 60 min of
chase, only 60% of CPY* was degraded. This final amount of CPY* is almost the same for the TmDM
(dark blue line), although it was degraded slower than in the Δder1 cells. Indeed, the degradation of
half of the CPY* present in the cell needed about 50 min. The degradation of this substrate for the
strain carrying the empty plasmid pRS415 (green line) was very slow and only 30% of the CPY* was
degraded at the end of the experiment. Those three strains, including the positive control, show a
strong ERAD defect, particularly the pRS415 strain.
The mutants tested showed a comparable behavior in every repetition of this experiment: a
strong ERAD defect for the TmDM, pRS415 and Δder1 cells, and a correct degradation for the S3A,
the T5A and the WT strains. However, the quantification in each of the assays resulted in a great
variability of the quantification values, when expressed as mean values. For this reason, the most
representative assays is presented in Figure 12. Another experiment is shown in Figure 15 (Annex
VIII.2).

48
Figure 12 - Degradation of CPY* in SBH1 mutant strains over time. (A) Cells were grown at 30°C to an OD600=2
in YPD medium. Cycloheximide was added to inhibit translation, and samples were taken every 15 min for 60
min. Whole-cell extracts were prepared and proteins were resolved on SDS-PAGE. They were transferred onto
nitrocellulose membranes and immunoblotted with CPY and Rpn12 as a loading control. (B) B) Quantification
of the experiment in (A). The analysis was performed using ImageJ.

49
3.2 - Combined mutants
In order to identify ERAD defects in the mutant strains (S3A/T5A; S3A+TmDM; T5A+TmDM
and S3A/T5A+TmDM) generated by Julien Simon, cycloheximide chase experiments were
performed as described in II.2.8. These strains (ref. II.1.3) were transformed with the plasmid
pDN431 carrying the gene encoding CPY*HA .
The results of this experiment are shown in Figure 14. A Δder1 mutant was used as a positive
control. This experiment could only be performed once due to time constraints.
Only 5 min after the beginning of the chase, half of the CPY* present in the S3A/T5A mutant
(blue line) and the S3A+TmDM (green line) was degraded. After 15 min, the amount of CPY* in the
S3A/T5A mutant started to increase, and finally reached a concentration of 20% at the end of the
chase, whereas the S3A+TmDM already reached its final degradation rate (80%) after 15 min and
stayed relatively constant until the end of the chase. The two strains with the higher CPY*
degradation rates are the T5A+TmDM (purple line) and the WT SBH1 (orange line) strains, with
85% and 95% of final degradation of CPY*, respectively. The T5A+TmDM showed a half life of CPY*
present of about 13 min, and the WT strain of 25 min. The latter degraded CPY* until it reached its
maximum rate after 45 min. The T5A+TmDM strain also degraded CPY* constantly until the end of
the chase. Those four strains, S3A/T5A, S3A+TmDM, T5A+TmDM and WT SBH1, did not show an
ERAD defect.
The first part of the graph is irregular for the following mutants, thus it will need more assays
to define it correctly. The TmDM (red line) reached a final CPY* degradation rate of 50% at the end
of the chase. The S3A/T5A+TmDM (light blue line) reached almost the same degradation rate, but
showed a small accumulation (10%) after 15 min of chase. 70% of the CPY* present at that time was
then degraded in 15 min and subsequently the rate stayed relatively stable. After 15 min of chase,
the pRS415 (dark blue line) transformant shows a very strong increase of CPY* (about 30%), probably
corresponding to a delay in the translocation of that protein into the lumen of the ER. During the
following 45 min of the chase, CPY* was degraded, and a final amount of 70% of CPY* was remaining
in the cell. In the Δder1 cells (burgundy-colored line), 20% of the present CPY* was degraded after
15 min, and the final degradation rate reached 30%. These four mutant strains, S3A/T5A+TmDM,
TmDM, pRS415 and Δder1 showed a strong ERAD defect, particularly the pRS415 strain and the Δder1
cells.
In order to confirm the previously obtained results, this experiment should be repeated.
4. TEST ON ANTIBODIES DIRECTED AGAINST SBH1P
Soromani et al. (2012) investigated the possible influence of phosphorylation of the cytosolic
domain of Sbh1p on its growth and on the translocation efficiency. Therefore, they performed site-
directed mutagenesis to mutate the presumable phosphorylation site to an unphosphorylable
amino-acid (Ala) and in particular, they investigated the phosphorylation on T5 position. Indeed, it
seems that this site and the two Pro residues that flank it have evolved independently in S. cerevisiae
and in mammals, and they are not present on Sbh2p.
To characterize this mutant (T5A), they performed a western blot, using Sbh1p antibodies
raised against the first 18 a.a. of the cytosolic domain of Sbh1p. The signal detected on the membrane
for the T5A mutant was reduced compare to the WT.

50
Figure 13 - Degradation of CPY* in combined SBH1 mutant strains over time. (A) Cells were grown at 30°C to
an OD600=2 in YPD medium. Cycloheximide was added to inhibit translation, and samples were taken every 15
min for 60 min. Whole-cell extracts were prepared and proteins were resolved on SDS-PAGE. They were
transferred onto nitrocellulose membranes and immunoblotted with antibodies directed against CPYp and
Rpn12p as a loading control. (B) Quantification of the experiment in (A). The analysis was performed using
ImageJ.
In order to verify the reliability of the antibody used, they performed another immunoblotting using
mutants lacking either SBH1 or SBH2. It was thus shown, that this antibody also recognizes Sbh2p,
even though it better recognizes Sbh1p. The only region that is different between both is comprised

51
between the second and the fifth a.a.. As the mutation tested is on the fifth a.a., it is possible that it
affected the interaction with the antibody, thus explaining the reduced signals.
A new Sbh1p antibody was raised against the a.a. 10 to 23 and a western blot was performed
on following strains in order to possibly confirm the previously formulated hypothesis. The WT
(SBH1SBH2) and the T5A mutated strain were used to verify if the latter also shows a reduced signal
if immunoblotted with another antibody. Δsbh1SBH2, SBH1Δsbh2 and Δsbh1Δsbh2 were used in
order to see if this newly raised antibody also recognizes Sbh2p.
The results obtained after immunoblotting with both antibodies (Sbh1 N-ter and Sbh1 10-23)
are presented in Figure 15. The WT strain was used as a positive and Δsbh1Δsbh2 as a negative
control. Figure 15 shows the results obtained with the antibody raised against the a.a. 1 to 18 (Sbh1
N-ter; A) and the results obtained using the antibody Sbh1 10-23 (B).
Figure 14 -Test on a new Sbh1p antibody and comparison with the previously used one. Cells were grown at 30°C
to an OD600=2, cell-extracts were prepared and proteins resolved on SDS-PAGE. After transfer on
nitrocellulose membrane, they were immunoblotted with both anti-Sbh1p antibodies. Anti-Sec61p directed
against its N-terminus (Sec61 N-ter) was used as loading control.
In (A), the bands detected for the WT and the T5A mutant strains are comparable to the
results described by Soromani et al. (2012): the T5A mutant shows a reduced signal compared to the
WT strain. The comparison between the very weak band detected in the Δsbh1SBH2 mutant and the
stronger one detected in the SBH1Δsbh2 mutant indicates that the antibody recognizes SBH1 better
than SBH2, thereby confirming the results obtained by Soromani et al.
In (B), an intense signal was detected for the T5A mutant, almost as strong as the one
detected for the WT. The figure also shows an intense signal for the SBH1Δsbh2 mutant, and a weak
one for Δsbh1SBH2.
After comparison with the results obtained by Soromani et al. (2012), the antibody directed
against the a.a. 10 to 23 seems to recognize the T5A mutant better than the antibody directed against
the N-terminus, and also to better detect Sbh1p. However, both antibodies detect a relatively
identical amount of Sbh2p, indicating that the detection for Sbh1p would be comparable for both
antibodies.

52
IV. Discussion
1. IMPORT OF GLUCOSIDASE I INTO THE ER
Soromani et al. (2012) discovered that T5 is an important phosphorylation site on Sbh1p but
if mutated to an unphosphorylable a.a. it doesn’t result in a hypophosphorylated protein, thus
suggesting that another site can be phosphorylated by the same kinase, such as the Ser at position
3. Another study, by Feng et al. (2007), demonstrated that a Sbh1 protein expressing only the TMD
was still functionally competent. Moreover they discovered that Δsbh1Δsbh2 cells presented a defect
in the translocation of Msn1, an enzyme that plays a role in the ERQC. Subsequently, in order to
characterize the function of the TMD, Zhao and Jäntti (2009) mutated randomly several a.a. in the
latter, leading to the discovery that the combination of two point mutations (P54S, V57G) resulted
in the inactivation of the TMD.
The aim of the previously described experiment was to investigate the import of Gls1,
another enzyme from the ERQC, in Δsbh1Δsbh2 cells carrying the phosphorylation mutations
mentioned above, and in a strain carrying the two point mutations in the TMD.
Almost 50% less Gls1 was imported in the strain carrying the WT SBH1 than in the WT strain,
but this can be easily explained by the fact that the WT strains carries both SBH1 and SBH2, whereas
the WT SBH1 was transformed in a Δsbh1Δsbh2 strains, and the cells are thus only expressing Sbh1p.
As it has been demonstrated that Sbh2p is part of the Ssh1 complex, that import proteins co-
translationally (Soromani et al., 2012), as does the Sec61 complex, when the gene encoding this
protein is deleted, it is likely that less proteins will be imported co-translationally.
Due to large variation of the Gls1 amount present in the cell depending on the experiments,
the results obtained for the S3A mutant are not significant, but the data indicate that an amount
comparable to the one manifested by the WT SBH1, was imported. Thus, it is possible to say that
this mutant doesn’t have a defect in the import of Gls1 in the ER. The same can be concluded for the
T5A mutant that shows a similar rate of Gls1 import.
The TmDM shows a stronger defect compared to the WT SBH1 control (about 50% less
import) and an even stronger one in regard to the WT strain (around 66%). The small error bar
indicates that for all performed experiments, the behavior is constant. This mutant thus presents a
defect in the import of Gls1 in the ER.
The TmDM used for almost all experiments was lacking both cytosolic and lumenal domains
of the protein and only expressed the TMD. Julien Simon generated a TmDM expressing the whole
Sbh1 protein, and it was used for the last experiment with these three mutants. The latter showed a
band for the TmDM, barely less intense than the WT strain. The results of only one experiment
cannot be very representative, nonetheless it might be possible that the mutations in the TMD of
the full protein do not have any impact on the import of Gls1 in the ER. This would mean that the
deletion of the cytosolic domain, however, induces import defects.

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