I only need help with part B. I don\'t know how to do this at all. You plan to u
ID: 168150 • Letter: I
Question
I only need help with part B. I don't know how to do this at all.
You plan to use a membrane trafficking assay that monitors the movement of a natural ly expressed extracellularly secreted protein (a-factor) through the secr etory pathway in budding yeast, Saccharomyces cerevisiae. This is a particularly useful experimental system because a-factor gets post-translationally processed in different organelles along the secretory pathway. Following co-translational transport into the endoplasmic reticulum (ER), a-factor is approximately 19KDa is glycosylated in the ER and increases to 26KDa, and it is then transported to the Golgi where it undergoes a proteolysis cleavage that results in the ture form of the protein 4KDa. The m ature a-factor is then transported to the plasma membrane for exocyt osis/secretion. Your goal is to identify which step in the secretory pathway is blocked in each of the uncharacterized ts-mutants based on the post-translational processing of a- factor. You grow samples of normal S. cerevisiae (SEC+) and four different ts-mutants (labeled MutA, MutB, MutC, and MutD) at 25 C for 1 day (the normal growth temperature for S e). The next day you keep some samples at 25°C while moving others o 36 C for 4 hours. You harvest the cells, and collect the prot lysates. You run these lysates on an SDS-PAGE gel, transfer the gel to a nitrocellulose membrane and do a Western blot with anti-a-factor prima antibody. The result of this Western blot is ry shown below: SEC+ MutA MutB MutC MutD 25 C 36 C 25 C 36 C 25 C 36 C 25 C 36 C 25 C 15Explanation / Answer
B. The four ts-mutants that are the result of temperature sensitive mutation are ts1189, ts942, ts817 and ts1100. At these four points of mutation, several mRNA exhibit defect and decay. Complementation of the growth and mRNA decay and defect, and the through the above-given experiment the genes that could be mutated in the four mutants are MRT4 gene at ts1189, GRC5 at ts942(encoding the L9 ribosomal protein), SLA2 at ts817(encoding a membrane protein) and THS1 at ts1100(encoding the threonyl t-RNA synthetase).