DEG15 PROCESSING OF CITRATE SYNTHASE 3 MUTANTS
Lydia Benitez , Kyle Helzer , Ali Dorchak , Laura Olsen
1
Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, GA
2
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI
,
ABSTRACT & INTRODUCTION:
Almost all eukaryotic cells contain small organelles known as peroxisomes.
Peroxisomal function depends on its protein makeup even though it does not
have the capability to synthesize its own. A family of proteins, known as per-
oxins, work with peroxisomal targetting signals (PTS) to transport the proper
proteins into the peroxisomal matrix. Citrate Synthase is a peroxisomal
enzyme which can be encoded by five different CSY genes. CSY1, CSY2, and
CSY3 are thought to contain PTS2s, and they are known to be peroxisomal.
PTS2 proteins usually undergo processing by a protease (DEG15 in plants and
TYSND1 in mammals), once they enter the lumen of the peroxisome, which
removes the PTS2 portion; however testing on CSY2 has shown that it is not
processed by DEG15. This experiment was designed to study the role of two
main peptides found within CSY3. Three mutants were created to make CSY3
resemble CSY1 and CSY2 at these same loci: CSY3 E29A showed 3.03% pro-
cessing, suggesting that the Glu is not a crucial component in the mecha-
nism. CSY3 R30P did show substantial decrease in percent processing (1.03%)
which suggests that the Arg is a key peptide in the DEG15 mechanism. CSY3
ER29-30AP results confirmed the hypothesis that the difference between
these these two peptides in CSY2 and CSY3 could be a cause for the lack of
processing seen in CSY2 but not in CSY3.
Figure 1: The figure above demonstrates how PTS containing proteins trans-
lated in the cytosol of the cell undergo a special mode of transportation
through the peroxisomal membrane. This transportation is performed mainly
by a group of proteins known as peroxins. There are two main types of PTSs,
PTS1 (C-terminus) and PTS2 (N-terminus). Pex5p is the transporter respon-
sible for binding PTS1 containing proteins and taking them to the peroxi-
somal membrane, while Pex7p holds the same responsibilities corresponding
to PTS2. Issues with these transport systems can result in disorders such as
Zellweger syndrome spectrum (ZSS) disorders. (Taken from Hayashi, M. and
Nishimura, M.)
CSY1: RLAVLNAHLTVSEPN---QVLPAIEPWCT”SAHITAAPHGSLKG 49.0 KDA
CSY2: RLAVLTAHLAVSDTVGLEQVLPAIAPWCT^SAHITAAPHGSLKG 52.5 KDA
CSY3: RLAVLSGHLSEGKQD-----SPAIERWCT^SADTSVAPLGSLKG 52.4 KDA
Figure 2: The figure above shows a piece of the alignment of the CSY amino
acid sequences. The red residues correspond to the PTS2 sequence and the
blue residues are similar to the known PTS2 consensus sequence. All of the
highlighted segments represent areas of interest--the yellow portion is under
investigation in this experiment. (Taken from 2. Helzer, K. and Olsen, L.)
INTERDISCIPLINARY
in the Structure and Function of Proteins
at the University of Michigan
1 2 2 2
METHODS:
The following mutants were designed using PCR site directed mutagenesis
CSY3 E29A, CSY3 R30P, and CSY3 ER29-30AP. They were expressed in
pCR®II-TOPO® plasmids, transformed and then the DNA was purified on a large-
scale. The DNA was then transcribed and translated in vitro using radioactive
35
S-methionine. DEG15 protease assays were incubated with the CSY3 proteins,
visualized using autoradiography, and quantified with a phospho-imager to d-
etermine percent processed
RESULTS & DISCUSSION:
Figure 3: The figure below (left) demonstrates what the linear form of CSY3 in
the pCR®II-TOPO® plasmid looks like. It is expected that this band should be
around 5.5 Kb in total because the gene itself 1.53 Kb while pCR®II-TOPO® is 4.0
Kb. The second band displays pCR®II-TOPO® once the gene had been dropped.
The top band (pCR®II-TOPO®) is right at the 4.0 Kb mark on the ladder, while the
lower band (CSY3) is between the 1.0 Kb and 1.65 Kb marks
5.0 Kb
4.0 Kb
5.0 Kb
1.65 Kb
1.0 Kb
1.0 Kb
Figure 4: The figure above (right) shows mRNA samples which were run on a gel
after the in vivo transcriptions. The higher bands are the linear form of the CSY3
genes in pCR®II-TOPO®. The lower bands are the mRNA product.
Figure 5: The figure above shows the results of both the translations and the
DEG15 protease assays. It is difficult to distinguish processing from this gel;
however, the values are represented by the percent processing found with
phospho-imaging. The processing labels are only true for the assay lanes.
Figure 6: The figure below shows that, as was expected, the CSY3 ER29-30AP
mutant (which most closely resembles CSY2) showed substantially less per-
cent processing (1.36%) by DEG15 than the wild type (5.88%) when tested
using an in vitro protease assay. The CSY3 E29A mutant showed slightly less
processing (3.03%), suggesting that the glutamate does not play a promi-
nent role in the mechanism while the CSY3 R30P mutant showed the great-
est decrease in processing (1.03%). This suggests that the arginine plays a
heavy role in the processing mechanism.
FUTURE RESEARCH:
Testing these results for reproducibility will give a better understanding of
the roles of these peptides in the DEG15 processing mechanism and allow
for a deeper understanding of protein transport into the peroxisome. This
knowledge will then be able to aid in future treatments for patients who face
many life-threatening peroxisome disorders.
REFERENCES:
1. Hayashi, M. and Nishimura, M. (2003) Entering a new era of research on
plant peroxisomes. Curr. Opin. Plant Biol. 6, 577–582.
2. Helzer, K. and Olsen, L. (2012) Processing of Citrate Synthase by the
Peroxisomal Protease DEG15 in Arabidopsis thaliana. MCDB Honors
Thesis University of Michigan. 28.
ACKNOWLEDGEMENTS:
I would like to thank the NSF for providing the funding for me to come to
Michigan, the College of Pharmacy IREU for every effort they put in into
making this program happen and pairing us with a primary investigator at
the University of Michigan, Dr. Laura Olsen for allowing me to work in her lab
and providing me with all materials necessary to do so, Kyle Helzer for shar-
ing his project with me and, along with Ali Dorchak, answered my many
questions through each day.