Date of Award

Spring 1-1-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Greg Odorizzi

Second Advisor

Gia Voeltz

Third Advisor

Jingshi Shen

Fourth Advisor

Tin Tin Su

Fifth Advisor

Rytis Prekeris

Abstract

Transmembrane proteins at the plasma membrane that are targeted for degradation in the lysosome are sorted through the endosomal pathway. At endosomes they are sorted into inward budding intralumenal vesicles (ILVs) through the concerted action of the endosomal sorting complexes required for transport (ESCRTs). Deletion of ESCRT genes in S. cerevisiae disrupts endosomal morphology leading to aberrant flattened stacks of endosomal membranes known as class E compartments. Recently we have shown that class E compartments form due to hyperactivation of the Rab5 homolog, Vps21, as well as failure to activate the Rab7 homolog, Ypt7. Here we show that class E compartment formation requires the guanine nucleotide exchange factor (GEF) Vps9. Disruption of ubiquitin binding by Vps9 severely diminishes localization of Vps9 at class E compartments but does not suppress the formation of class E compartments. In addition we find the removal of Vps9 from endosomes requires the putative endosomal conversion factor Mon1-Ccz1 as well as endosome-to-vacuole fusion. Mon1-Ccz1 fails to localize to class E compartments but loss of Mon1-Ccz1 does not disrupt MVB biogenesis indicating ESCRT activity precedes Rab conversion. Our data suggest a model where endosomal Rab conversion occurs after completion of ESCRT-mediated sorting and involves multiple mechanisms including removal of ubiquitin by ESCRTs and recruitment of Mon1-Ccz1.

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Cell Biology Commons

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