Please help with this, I don\'t even know where to start. You plan to use a memb
ID: 168149 • Letter: P
Question
Please help with this, I don't even know where to start.
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
In mutA the protein size is larger than 4 KDa which is suggesting improper proteolysis cleavage step. In mutB, various protein sizes observed which are larger than 26KD. The sizes can increase by glycosylation. That is suggesting the defective process of glycosylation in Endoplasmic reticulum. In mutC, the protein did not synthesized properly because size is nearly to 19KD. Another defection of glycosylation found in MutD.