Protein targeting or protein sorting is the biological mechanism by which proteins are transported to their appropriate destinations in the cell or outside it. Proteins can be targeted to the inner space of an organelle, different intracellular membranes, plasma membrane, or to exterior of the cell via secretion.

1. PRESENTED BY :-
ANISHA MUKHERJEE
M.Sc SECOND SEMESTER
BIOTECHNOLOGY
GUIDED BY :-
Mrs. DIVYA PAIKARA
MA’AM

2. INDEX
 PROTEIN TARGETING
 TARGETING PATHWAYS
● Post-translational Targeting
● Co-translational Targeting
 ROLE OF RIBOSOMES IN PROTEIN TARGETING
 TARGETING SIGNALS
• Types of Targeting Peptides
 Pre-sequences
 Internal Targeting Peptides
 COMPARTMENTAL TRANSLOCATION OF PROTEIN
• Gated Transport
 Protein Transport into the nucleus
• Trans-membrane Transport
 Protein Transport into the Mitochondria
 Protein Transport into the Chloroplast
 Protein Transport into the Peroxisome
• Vesicular Transport
 Cells Import Proteins by Receptor-Mediated Endocytosis
 REFERENCES

3. PROTEIN TARGETING
 The newly synthesized proteins by the cell are sorted and then
transported to their correct destination so that they can carry out
appropriate function. The process is known as protein targeting.
 Proteins can be targeted to the inner space of an organelle,
different intracellular membranes, plasma membrane or to
exterior to the cell via secretion.
 This delivery process is carried out based on information
contained in the protein itself.
 Correct sorting is crucial for the cell; errors can lead to diseases.

4. Fig. A Typical Cell

5.  Protein has to be correctly localized to perform proper functions.
E.g. :- Receptors- Plasma Membrane
DNA polymerase- Nucleus
Catalase – Peroxisomes
Insulin- outside
 All proteins begin to be synthesized on cytosolic ribosomes.
 Sorting or translocation can occur :- co-translational and post-translational.
 If the protein is for cytosolic functions, the synthesis will be finished on free ribosomes
and the peptide is released into the cytosol.
 If the protein is destined for nucleus, mitochondria or peroxisomes the synthesis is also
finished on cytoplasmic ribosomes and the peptide is released to the cytosol (to be sorted
later or post-translationally).
 If the protein is going to be secreted from the cell or it is destined for the membranes, the
ribosome with the nascent peptide is targeted to the ER (ER becomes rough) and sorting
is done during translation (co-translationally).
PROTEIN TARGETING (Cont.)

6. TARGETING PATHWAYS
 Post-translational
Targeting :-
•Nucleus
•Mitochondria
•Peroxisomes
 Co-translational
Targeting
(secretory pathway) :-
•ER
•Golgi
•Lysosomes
•Plasma membrane
•Secreted proteins

8. POST-TRANSLATIONAL TRANSLOCATION
Post-translational translocation is the pathway which
occurs after the process of translation.
Even though most proteins are co-translationally
translocated, some are translated in the cytosol and
later transported to their destination. This occurs for
proteins that go to mitochondrion, a chloroplast, or a
peroxisome.

9. CO-TRANSLATIONAL TRANSLOCATION
In this pathway, transport of protein occurs during
translation which is not completed fully.
 Synthesized protein is transferred to an SRP receptor
on the endoplasmic reticulum (ER), a membrane
enclosed organelle. There, the nascent protein is
inserted into the translocation complex.

10. ROLE OF RIBOSOMES IN PROTEIN TARGETING
 Two categories of ribosomes have been identified :-
 Those that remain free in the cytosol.
 Those that remain bounded to the Endoplasmic Reticulum.
o The ER devoid of ribosomes is called Smooth Endoplasmic Reticulum
(SER).
o The membrane bound ribosomes synthesize secretory proteins, lysosomal
proteins & proteins that span the plasma membrane.
o The basic difference between membrane bound & free ribosomes is that
the signaling sequences in the nascent proteins direct the membrane bound
ribosomes to become attached to the ER.
o Once the secretory proteins are synthesized by the ribosomes bound to the
rough endoplasmic reticulum (RER), they are translocated into the lumen of
RER where they undergo folding to assume final conformation.

11. TARGETING SIGNALS
Targeting signals are the pieces of information that
enable the cellular transport machinery to correctly
position a protein inside or outside the cell.
This information is contained in the polypeptide chain or
in the folded protein.
In the absence of targeting signals, a protein will remain
in the cytoplasm.

12. TYPES OF TARGETING PEPTIDES
 The continuous stretch of amino acid residues in the chain
that enables targeting are called signal peptides or
targeting peptides.
 There are two types of targeting peptides :-
1. The pre-sequences
2. The internal targeting peptides

13. 1. PRESEQUENCES
 The pre-sequences of the targeting peptides are often found at the N-
terminal extension but in case of peroxisomes the targeting sequence is on
the C-terminal extension mostly.
 Signal sequence is a short peptide (usually 16-30 amino acids long)
present at the N-terminus of the majority of newly synthesized proteins that
are destined towards the secretory pathway.
 It is composed of between 6-136 basic and hydrophobic amino acids.
 Signal sequences are removed from the finished protein by specialized
signal peptidases once the sorting process has been completed.

14. 2. INTERNAL TARGETING PEPTIDES
 The targeting peptides are often found at within the polypeptide chain,
not at any end .

15. COMPARTMENTAL TRANSLOCATION OF PROTEINS
 There are three types of transport of
proteins through different
compartments of cell :-
a) Gated transport (Nucleus)
b) Trans-membrane transport (Mitochondria,
Peroxisomes, chloroplast)
c) Vesicular transport (E.R, Clathrin mediated
endocytosis)

16. GATED TRANSPORT
 The protein transfer is from or to the
nucleus and is aided by nuclear pore.
 The nuclear pore complexes function as
selective gates that actively transport
(with expenditure of energy) specific
macromolecules and macromolecular
assemblies.

17. PROTEIN TRANSPORT INTO THE NUCLEUS
 The nuclear envelope encloses the DNA and defines the nuclear
compartment. This envelope consists of two concentric membranes that are
penetrated by nuclear pore complexes.
 The inner nuclear membrane contains specific proteins that act as binding
sites for chromatin and for the protein meshwork of the nuclear lamina that
provides structural support for this membrane.
 The inner membrane is surrounded by the outer nuclear membrane, which is
continuous with the membrane of the ER. Like the membrane of the ER the
outer nuclear membrane is studded with ribosomes engaged in protein
synthesis .
 The proteins made on these ribosomes are transported into the space
between the inner and outer nuclear membranes (the perinuclear space),
which is continuous with the ER lumen, with ribosomes engaged in protein
synthesis.
 Many proteins,histones, DNA and RNA polymerases, gene regulatory
imported into the nuclear compartment from the cytosol. Proteins and RNA-
processing proteins, selectively tRNAs and mRNAs are synthesized in the
nuclear compartment and then exported to the cytosol.

18. IMPORT AND EXPORT OF PROTEINS TO NUCLEUS
 The transport is bidirectional and occurs through the nuclear pore
complexes (NPCs). These are complex structures composed of
aggregates of about 30 different proteins.
 The nuclear envelope has hundreds of NPCs, located where the
two nuclear membranes meet.
 NPC is made of three types of nucleoporins :-
1. Structural nucleoporins
2. Membrane nucleoporins
3. FG nucleoporins.
 Each NPC has multiple copies of at least 30 different proteins
called nucleoporins.
 Most polypeptides destined for the nucleus have address labels,
called nuclear localization signals (NLSs), consisting of one or
more short internal sequences with basic amino acids.
 Importins and Ran (a monomeric G‐protein that can exist in either
the GTP‐bound or GDP‐bound conformation) help in import of
proteins containing NLS.
 Proteins similar to importins, referred to as exportins, are involved
in the export of many macromolecules (various proteins, tRNA
molecules, ribosomal subunits and certain mRNA molecules) from
the nucleus. Cargo molecules for export carry nuclear export
signals (NESs).
 The family of importins and exportins are referred to as
karyopherins.

19. MECHANISM
 Import of proteins containing the NLS sequence requires a nuclear transport receptor known
as importin. These free importins in the cytoplasm binds to their cognate NLS in a cargo
protein, forming a importin-cargo complex.
 The importin-cargo complex then binds to the FG repeats of FG nucleoporins which allows it
to enter into the nucleoplasm. There the importin interacts with Ran.GTP, which causes a
conformational change in it. So that the cargo protein gets disassembled from the importin-
cargo complex in the nucleoplasm.
 The importin Ran⋅GTP complex then diffuses back through the NPC to the cytoplasm. Then
Ran interacts with a specific GTPase activating protein (Ran-GAP).
 This interaction stimulates Ran to hydrolyze its bound GTP to GDP, which causes it to
convert to a conformation that has low affinity for importin, so that the importin is released into
the cytoplasm, where it can participate in another cycle of import.
 Ran⋅GDP travels back through the pore to the nucleoplasm, where it encounters a specific
guanine nucleotide exchange factor (Ran-GEF) that causes Ran to release its bound GDP in
favor of GTP.

20. Fig. Mechanism for nuclear import of proteins.

21. TRANSMEMBRANE TRANSPORT
 Membrane-bound protein translocators directly
transport specific proteins across a membrane
from the cytosol into a space that is
topologically distinct.
 The transported protein molecule usually must
unfold to snake through the translocator .
 The initial transport of selected proteins from
the cytosol into the ER lumen or from the
cytosol into mitochondria.

22. PROTEIN TRANSPORT INTO THE MITOCHONDRIA
 There are four locations inside the
mitochondria to which proteins are
translocated. They are:-
1. Outer membrane.
2. Inner membrane
3. Inter membranal space
4. Mitochondrial matrix

23. PROTEIN TRANSLOCATORS IN THE MITOCHONDRIAL MEMBRANES
• These complexes contain some components
that act as receptors for mitochondrial precursor
proteins and other components that form the
translocation channel :-
 The TOM complex- It transports mitochondrial
precursor proteins, nucleus encoded mitochondrial
proteins.
 The TIM23 complex- It transports proteins into
the matrix space.
 The TIM22 complex- It transports mediates the
insertion of a subclass of inner membrane proteins,
including the carrier protein that transports ADP,
ATP, and phosphate.
 The OXA complex- mediates the insertion of
inner membrane proteins .

24. MECHANISM
 Most mitochondrial proteins are synthesized as cytosolic precursors containing uptake peptide signals.
 Cytosolic chaperones deliver preproteins to channel linked receptors in the mitochondrial membrane.
 The preprotein with presequence targeted for the mitochondria is bound by receptors and the General Import Pore (GIP) (Receptors
and GIP are collectively known as Translocase of Outer Membrane or TOM) at the outer membrane.
 Three mitochondrial outer membrane receptors are known- TOM20, TOM22 and TOM70.
• TOM70: Binds to internal targeting peptides and acts as a docking point for cytosolic chaperones.
• TOM20: Binds presequences
• TOM22: Binds both presequences and internal targeting peptides
• The TOM channel (TOM40) is a cation specific high conductance channel with a molecular weight of 410 kDa and a pore diameter
of 21A.
o Proteins are transferred to TOM40 pore protein & translocated across the outer membrane.
o The preprotein is translocated through TOM as hairpin loops.
o The proteins are then transferred to a second protein complex in the inner membrane(Tim23).
 The presequence translocase23 (TIM23) is localized to the mitochondrial inner membrane and acts as a pore forming protein which
binds precursor proteins with its N-terminus.
 TIM23 acts as a translocator for preproteins for the mitochondrial matrix, the inner mitochondrial membrane as well as for the
intermembrane space.
 There are two Tim complexes – Tim22 and Tim23 complex.
 The Tim23 complex is formed by the three essential inner membrane proteins: Tim50 (with a receptor function in intermembrane
space), Tim23 (channel-forming protein) and Tim17 (involved in motor recruitment).
 The Tim22 contains inner membrane proteins Tim18, Tim22 and Tim54.

25.  TIM50 is bound to TIM23 at the inner mitochondrial side and found to bind presequences.
 TIM44 is bound on the matrix side and found binding to mtHsp70.
 The presequence translocase22 (TIM22) binds preproteins exclusively bound for the inner
mitochondrial membrane.
 Mitochondrial matrix proteins are then translocated across the inner membrane through Tim23.
 Translocation into the matrix thus occurs at “contact sites” where the outer and inner
membranes are in close proximity.
 Soon after the N-terminal matrix-targeting sequence of a protein enters the mitochondrial matrix,
it is cleaved off by a protease (mtHsp 70)that resides within the matrix.
 The emerging protein is also bound by matrix Hsp70, a chaperone that is localized near the
translocation channels in the inner mitochondrial membrane by interaction with transmembrane
protein Tim44.
 This binding stimulates ATP hydrolysis by matrix.
 Hsp70, and together, Tim44 and Hsp70 are thought to power translocation of proteins into the
matrix.
 Final folding of many proteins requires chaperonins present in the mitochondrial matrix.

27.  Protein transport into Inner Membrane or Inner Membrane Space requires 2 signal sequences :-
1. Second signal =hydrophobic sequence; immediately after 1st signal sequence.
2. Cleavage of N-terminal sequence unmasks 2nd signal used to translocate protein from matrix into or across Inner Membrane using
OXA.
3. OXA also used to transport proteins encoded in mitochondria into Inner Membrane.
4. Alternative route by passes matrix; hydrophobic signal sequence = “stop transfer”.

28. ATP HYDROLYSIS AND A H+ GRADIENT ARE USED TO DRIVE PROTEIN
IMPORT INTO MITOCHONDRIA
 Mitochondrial protein import is fueled by ATP hydrolysis at two discrete sites,
one outside the mitochondria and one in the matrix .
 In addition, another energy source is required: an electrochemical H+ gradient
across the inner mitochondrial membrane.
 The requirement for hsp70 and ATP in the cytosol can be bypassed if the
precursor protein is artificially unfolded prior to adding it to purified mitochondria.

29. REPEATED CYCLES OF ATP HYDROLYSIS BY MITOCHONDRIAL
HSP70 COMPLETE THE IMPORT PROCESS
 Thermal ratchet model :-
The emerging chain slides back and forth in the TIM23
translocation channel by thermal motion. Each time a
sufficiently long portion of the chain is exposed in the
matrix, an hsp70 molecule binds to it, preventing further
backsliding and thereby making the movement
directional. Thus, a hand-over-hand binding of multiple
hsp70 proteins translocates the polypeptide chain into
the matrix.
 Cross-bridge ratchet model :-
The hsp70 proteins that bind to the emerging
polypeptide chain undergo a conformational change,
driven by ATP hydrolysis, that actively pulls a segment of
the polypeptide chain into the matrix. A new hsp70
molecule can then bind to the segment just pulled in and
repeat the cycle.

30. PROTEIN TRANSPORT INTO THE CHLOROPLAST
 In chloroplast, the targeting signal is
correspondent to Transit peptide(TP).
 The preprotein for chloroplasts may contain a
stromal import sequence or a stromal and
thylakoid targeting sequence.
 The majority of preproteins are translocated
through the Toc and Tic complexes located
within the chloroplast envelope.
 The signal sequence (transit peptide) binds with
target protein along with chaperone cytosolic
Hsp70. This is the signal to move the
polypeptide through Toc complex.
 Stromal peptidase cleave the target sequence
and pull the rest of polypeptide inside.
Fig. A Choloroplast

31. TRANSLOCATION OF PROTEIN IN CHLOROPLAST
 The vast majority of chloroplast proteins are
synthesized as precursor proteins (preproteins) in the
cytosol and are imported post-translationally into the
organelle.
 Preproteins that contain a cleavable transit peptide are
recognized in a GTP-regulated manner by receptors of
the outer-envelope translocon, which is called the TOC
complex.
 The preproteins cross the outer envelope through an
aqueous pore and are then transferred to the
translocon in the inner envelope , which is called the
TIC complex.
 The TOC and TIC translocons function together during
the translocation process.
 Completion of import requires energy, which probably
comes from the ATP-dependent functioning of
molecular chaperones in the stroma.
 The stromal processing peptidase then cleaves the
transit sequence to produce the mature form of the
protein, which can fold into its native form.

32. PROTEIN TRANSPORT INTO THE PEROXISOMES
 Peroxisomes are small organelles bounded by a single
membrane. All enzymes found in peroxisomes are
synthesized in the cytosol.
Examples:- Catalases, Urate oxidases.
 Peroxisomal targeting sequences are needed for the
import of proteins into peroxisomal matrix. They are as
follows:
a) PTS 1 :-
 Most of the peroxisomal matrix proteins have this
sequence. The sequence consists of Ser-Lys-Leu at
the C-terminus.
Example- Catalases.
b) PTS 2 :-
Very few peroxisomal proteins have this sequence at
N-terminus.
Example- Thiolases.
Fig. Structure of peroxisome.

33. MECHANISM OF PROTEIN SORTING IN PEROXISOMES
 In the cytosol, PTS1 binds to a receptor called Pex5. It has the ability to switch from a
monomeric form to an oligomeric form which is then embedded in a complex protein called
Pex14 in the peroxisomal membrane.
 Pex14 adjusts itself according to the size of the PTS1-bearing cargo molecules.
 Once the PTS1-bearing cargo molecule is released into the interior of the peroxisome, the
oligomeric complex of Pex5 and Pex14 is actively disassembled, thus releasing Pex5 back
into the cytoplasm in a soluble state.
 The peroxisome import machinery translocates folded proteins across the membrane.
 Pex5 recycling involves modification of membrane-bound Pex5 by ubiquitinylation. A
complex of the peroxisomal membrane proteins Pex10, Pex12 and Pex2 transfers a ubiquitin
moiety to Pex5.
 Pex1 and Pex6 anchored to the peroxisomal membrane by Pex15, recognize ubiquitinylated
Pex5 and remove it from the oligomeric complex with Pex14 with the help of ATP hydrolysis
thereby releasing it into the cytosol.
 After the removal, cytosolic Pex5 is ready to carry out another cycle of binding to a PTS1-
bearing protein.
 Target sequences are not removed in the matrix.

34. Fig. PTS1-directed import of peroxisomal matrix proteins.

35. VESICULAR TRANSPORT
 Proteins from the ER to the Golgi apparatus
and proteins to E.R, clathrin mediated-
endocytosis (CME), for example, occurs in
this way.
 Transport intermediates— which may be
small, spherical transport vesicles or larger,
irregularly shaped organelle fragments—
ferry proteins from one compartment to
another.
 The transfer of soluble recognized by a
complementary receptor in the appropriate
membrane.

36. CELLS IMPORT PROTEINS BY RECEPTOR- MEDIATED ENDOCYTOSIS
 Some proteins are imported into cells from the surrounding medium; examples in eukaryotes include
Low density lipoprotein (LDL), the iron-carrying protein transferrin, peptide hormones, and circulating
proteins destined for degradation.
 The proteins bind to receptors in invaginations of the membrane called coated pits, which concentrate
endocytic receptors in preference to other cell-surface proteins.
 The pits are coated on their cytosolic side with a lattice of the protein clathrin, which forms closed
polyhedral structures.
 The clathrin lattice grows as more receptors are occupied by target proteins, until a complete
membrane-bounded endocytic vesicle buds off the plasma membrane and enters the cytoplasm.
 The clathrin is quickly removed by uncoating enzymes, and the vesicle fuses with an endosome.
 ATPase activity in the endosomal membranes reduces the pH therein, facilitating dissociation of
receptors from their target proteins.
 The imported proteins and receptors then go their separate ways, their fates varying with the cell and
protein type.
 Transferrin and its receptor are eventually recycled.
 Some hormones, growth factors, and immune complexes, after eliciting the appropriate cellular
response, are degraded along with their receptors.
 LDL is degraded after the associated cholesterol has been delivered to its destination, but the LDL
receptor is recycled.
 Receptor-mediated endocytosis is exploited by some toxins and viruses to gain entry to cells.
Influenza virus, diphtheria toxin, and cholera toxin all enter cells in this way.

37. Fig. Clathrin mediated endocytosis

38. REFERENCES
LEHNINGER :- Principles of Biochemistry ; Cox and Nelson ;5th
edition
 Molecular cell Biology:- Lodish and Berk et al ; 5th edition
 www.slideshare.net
 www.wikipedia.org