1. VDAC4 and TGrPE1 Protein Localization
and Gene Expression in Tetrahymena Thermophilia
George Rizk, Sarabjeet Seehra, and Douglas Chalker, Ph.D
Tetrahymena thermophilia is good eukaryotic model organism to use to
better understand processes such as protein translation and apoptotic-
pathways in mitochondria.
Two genes in the Tetrahymena genome, called Tetrahymena GrPE 1
(TGrPE1) and voltage dependent anion selective channel 4 (VDAC4) may
have important roles in such mitochondrial processes. TGrPE1 encodes for a
protein product, whose eukaryotic orthologs facilitate the exchange of ADP
for ATP on proteins involved in proper protein folding, such as Hsp70.
VDAC4 encodes for a porin trans-membrane protein, whose orthologs, such
as VDAC1 and Tom40, are involved in communication among organelles and
trans-membrane transport. Evidence gathered from bioinformatics analysis
and protein localization studies support VDAC4 and TGrPE1 involvement in
mitochondrial processesThThe goal of the study is to determine the localization
and possible functions of these proteins.
Acknowledgements
Generation of YFP Transformants
Background VDAC4 Localizes to the Mitochondria & Basal
Bodies
Colocalization of TGrPE1 and
VDAC4
Conclusions and Future Directions
TGrPE1 Localizes to the Mitochondria
• VDAC4 exhibits clear localization to mitochondria, likely the outer membrane.
Mitochondrial staining suggests VDAC4 localizes to the cilliary basal bodies.
As a porin, VDAC4 could have intra-flagellar transport properties, acting as an
interface between the mitochondria and cilia to facilitate cellular metabolism
and movement. VDAC4 may be involved in the regulation of ciliogenesis.
Sophisticated VDAC4 purification experiments determine channel size and
pore dimensions. Studying the transport activity through bilayer reconstitution
could measure ion flow, labeled molecule monitoring, and voltage-
conformation correlations.
 TGrPE1 is a protein that localizes at or near mitochondria of Tetrahymena, as
shown by the YFP fusion localization, mitochondrial staining, and co-
localization of TGrPE1 with VDAC4. The presence of a GrPE1 domain in the
protein product of TGrPE1 suggests that it may be involved in the regulation of
chaperone proteins in Tetrahymena by ATP/ADP exchange. Future
experiments, such as yeast-two hybrid experiments with chaperone proteins in
Tetrahymena, will focus on determining if TGrPE1 plays a role in protein
folding and regulation.
The GOI was directionally cloned into a pENTR-
D Topo vector by strand invasion. The pENTR-
GOI vector verified by restriction enzyme digest
and transformed into E coli
The GOI replaced the gateway cassette of the
pICY-gtw vector by a lambda recombinase (LR)
reaction, and verified by restriction enzyme
digests.
The pICY-GOI
vector was
transformed into
Tetrahymena
thermophilia by
electroporation
A B
Figure 1: VDAC4 protein localization by YFP in growing Tetrahymena cells
A) VDAC4 localizes to the mitochondria along the longitudinal cilliary rows of the cell. B) Bright field
image corresponding to photo on left as reference for cellular structures.
Figure 2: localization in conjugating
Tetrahymena VDAC4 localizes to the cilliary
basal bodies underneath and along the cilia of
the cell.
Figure 3: Mitochondrial stain of growing Tetrahymena
Red mitochondrial stain highlighting the mitochondria.
Confirms presence of VDAC4 localization outside of
mitochondria in left photo
We would like to thank Anna Ballard and Andrew Jezewski for their help on this project.
Special thanks to D.L. Chalker, National Science Foundation, and Howard Hughes
Medical institute.
A B
Figure 4: TGrPE1 protein localization by YFP in growing Tetrahymena cells
A) TGrPE1 localizes near basal bodies and is scattered throughout the Tetrahymena, indicative of
mitochondrial localization. B) Bright field image provided as a reference for cellular topology.