Download Determining Compensatory Genes from Loss of Vacuolar

Determining Compensatory Mechanisms for Loss of Vacuolar Function in
Saccharomyces cerevisiae
Nick Netzel*, Jillian Wisby*, Pamela A. Marshall. Arizona State University at the West Campus, Glendale, AZ.
*students contributed equally to this work
Table 1: Up-regulated Genes of Interest
Abstract
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Saccharomyces cerevisiae is dependent on its vacuole for proper sorting, degradation, and
recycling of proteins, organelles, and other biomolecules that occur in the cell. For these
processes to function properly, vacuolar docking from incoming vesicles must be achieved. Yeast
mutants that have a characteristic loss of function vacuole are classified as vps mutants. The
current study utilizes two similar vps mutants, vps33 and vps41, both of which are able to
survive with a loss of function vacuole under laboratory conditions. The purpose of the current
study is to compare these vps mutants via DNA microarray analysis and determine if similar
genes are being expressed. Thus far, analysis has revealed vps33 and vps41 expressing similar
compensatory genes which include four genes of interest (YLR243w being the most interesting),
five genes resulting from stress, and three genes involved in mitochondrial activity. Due to
YLR243W’s level of up-regulation in the mutants and its essential role in actin cytoskeleton
formation, this gene will be a central focus in future studies.
wt
vps33 vps41
Table 2: Genes Associated with Stress Induced up-regulation
Figure 2: Gel
Confirming
Successful RNA
Extraction
Introduction
Cells must have specific ways to transport, store, and breakdown biological molecules. In
humans, there are many DNA mutations that lead to malfunctions in these cellular transport
processes and disease states. To better understand these processes we use a model organism,
the yeast Saccharomyces cerevisiae. Central to S. cerevisiae’s protein sorting, storing and
biomolecular breakdown is the vacuole. This characteristic vacuole is responsible for the major
pathway in which degradation and recycling of proteins and other organelles occurs; the other
pathway involves the peroxisome [1]. In comparison, human cells also have a peroxisome, but
the lysosome is involved in the major pathway responsible for biomolecular breakdown [4].
Unlike human cells, yeast is easy and inexpensive to grow but can still yield important insights
into human disease. In the laboratory, strains of yeast have been cultured which contain loss of
function vacuoles yet still sustain the ability to survive. Yeast strains such as these are classified
as vps mutants [2]. The specific mutants used in this study are vps33 and vps41 (also known as
YLR396 and YDR080, respectively). Both mutant types prevent endosome vesicle docking to the
yeast vacuole [6] (Figure 1).
By taking advantage of the yeast vacuole and human lysosome relationship, improved treatment
plans can be made for individuals suffering from lysosomal storage disorders. Lysosomal storage
disorders are genetically inherited as the result of a defect in lysosomal proteins or a defect in
protein transport to the lysosome. These disorders represent a group of at least 41 distinct
genetic diseases [5]. The most common lysosomal storage disorder is Gaucher’s disease, which
affects 1 in 57,000 births and is characterized by a deficiency of the enzyme glucocerebrosidase,
causing fatty tissue to build up in vital organs such as the spleen and liver [3]. Symptoms of
this and other lysosomal storage disorders (Tay-Sachs and Krabbe disease) include an enlarged
spleen and liver, liver malfunction, skeletal disorders, and bone lesions.
Results/ Discussion
Figure 3: DNA Microarrays of
vps33 (left) and vps41 (right).
Our hypothesis is that changes in gene expression compensate for the loss of vacuolar function
in vps33 and vps41. We used DNA microarray technology to determine the changes in gene
expression that allow mutant strains lacking vacuole function to thrive in the lab.
We have successfully compared the complete genomes of two different
mutant strains of Saccharomyces cerevisiae, vsp33 and vsp41, to a wild
type strain using DNA microarray technology. Repeated trials have
consistently shown up-regulation of 12 different genes in both mutant
strains (Tables 1 & 2). Analysis using the S. cerevisiae database has
shown 4 of the selected genes to be be of possible significance in genetic
compensation for the loss of vacuole function. The most interesting upregulation is YLR243W. As it turns out, this gene’s protein product
interacts with other proteins that play a part in actin cytoskeleton
formation, allowing for regulation of endocytosis during times of osmotic
stress [7]. At this point it is hypothesized that the other 8 genes under
consideration are likely linked to stress induced up-regulation due to
experimental growth conditions. Further studies should investigate the
relationship between YLR243W up-regulation and actin cytoskeleton
formation. Future studies will include increased cellular stress by the
addition of 100 and 200 mM calcium. Since the yeast vacuole is a
primary storage center for divalent cations, we expect to see gene
compensation more definitively.
Literature Cited
1. Bryant, N.J., and Stevens, T.H. 1998. Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to
the yeast vacuole. Microbiology and Molecular Biology Reviews 62: 240-247.
Materials and Methods
Vacuole
X
Nucleus
Autophagy
Golgi
ER
Endosome
Secretory
Vesicle
Figure 1: Biomolecular Sorting, Storage, and
Breakdown in Yeast. Both vps33 and vps41are
not able to bind incoming vesicles to the vacuole.
The red “X” indicates where vps mutants are
hindered which leads to a total loss of vacuolar
function in cell strains lacking these genes.
-RNA Extraction
S. cerevisiae RNA was extracted using Ambion’s RiboPureYeast Kit and purified using Qiagen’s PCR Purification Kit
-Gel Electrophoresis
Agarose gel electrophoresis was executed to visually ensure
RNA was extracted (Figure 2)
-Microarray Preparation
DNA microarrays were produced using a Genisphere 3DNA
Array 350 Labeling Kit (Figure 3)
-Analysis
Initial DNA microarray analysis was performed utilizing
MicroArray Genome Imaging and Clustering Tool (MAGIC Tool)
Up-regulated genes were then cross referenced with the
Saccharomyces Genome Database (Tables 1 and 2) [7]
2. Herman, P.K., and Emr, S.D. 1990. Characterization of VPS34, a gene required for vacuolar protein sorting and
vacuole segregation in Saccharomyces cerevisiae. Molecular and Cellular Biology 10: 6742-6754.
3. Hollak, C.E., van Weely, S., van Oers, M.H., and Aerts, J.M. Marked elevation of plasma chitotriosidase activity: a
novel hallmark of Gaucher disease. Journal of Clinical Investigation 93: 1288-1292.
4. Luzio, J., Poupon, V., Lindsay, M., Mullock, Piper, R., and Pryor, P. 2003. Membrane dynamics and the biogenesis of
lysosomes. Molecular Membrane Biology 20: 141-154.
5. Meikle, P.J., Hopwood, J.J., Clague, A.E., and Carey, W.F. 1999. Prevalence of lysosomal storage
the American Medical Association 281: 249-254.
disorders. Journal of
6. Rieder, S., and Emr, S.D. A novel RING finger protein complex essential for a late step in protein transport to the yeast
vacuole. Molecular Biology of the Cell 8: 2307-2327.
7. Saccharomyces Genome Database <http://www.yeastgenome.org/>
Acknowledgements
NN was supported by the SRP Life Sciences Scholarship. Special thanks to the Genome Consortium for
Active Teaching for training (PAM), support, and subsidized micrarrays.