PhD Oral Exam - Thomas David Daniel Kazmirchuk, Biology

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Lysosomes are key mediators of cell metabolism which require nutrient transporters to function. To maintain homeostasis, these transporters are selectively degraded by lysosomal degradation pathways including the Vacuolar Recycling and Degradation (VRED), microautophagy, and Intralumenal Fragment (ILF) pathways. To promote selectivity, ubiquitin is thought to label VRED and microautophagy cargos by recruiting E3-ligases and adaptor proteins. The label responsible for ILF pathway selectivity remains elusive. In this thesis I use Saccharomyces cerevisiae and its vacuolar lysosome as models to elucidate the label for the ILF pathway by studying three proteins: the iron oxidase Fet5, the adaptor protein Ssh4, and the methionine permease Mup1.

I first examined Fet5 and find that it is not ubiquitylated when triggered for degradation by cycloheximide (CHX, TOR-kinase activation) based on immunopurification. I then assessed whether mutating Fet5 to prevent ubiquitylation impacts degradation by exchanging the only lysine residue in the cytosolic carboxyl-tail (C-tail) to an arginine (Fet5K603R) or by removing the entire C-tail (Fet5-tail) to block association of labeling machinery. By tracking GFP-tagged versions of these proteins by microscopy and Western blot, I find that Fet5K603R degradation by the ILF pathway persists after CHX treatment, suggesting this lysine and potential ubiquitylation are not required. Degradation of Fet5C-tail is blocked implicating the C-tail in stimulus-specific degradation.

I next studied Ssh4 as this adaptor has been implicated in the degradation of vacuolar transporters. I find that deleting SSH4 prevents CHX-triggered degradation of Fet5. Furthermore, Ssh4 is ubiquitylated and sorted into the ILF pathway. Together, these results suggest that ubiquitylated Ssh4 may mediate stimulus-specific degradation of Fet5.

Finally, I assessed whether Mup1 can be selectively degraded by the ILF pathway. Mup1 is ubiquitylated by ESCRTs and serves as a model for direct-ubiquitylation. I confirm that Mup1 is degraded by the ILF pathway after methionine addition when ESCRTs are deleted suggesting that the ILF pathway may recognize cargo proteins by direct-ubiquitylation.

The results presented in this thesis challenge the dogma that selective degradation of vacuolar nutrient transporters relies solely on direct-ubiquitylation by demonstrating that the labeling of ILF cargos can involve the recruitment of ubiquitylated adaptors to promote indirect-ubiquitylation.