Date of Award
Doctor of Philosophy (PhD)
The sequential recruitment and assembly of endosomal sorting complexes required for transport (ESCRTs) at the endosomal membrane mediate the selection and clustering of cargoes into vesicles that bud into the lumen of the endosome. In addition to regulating this sorting process at endosomes, in mammalian cells ESCRTs are additionally required for the budding of many types of enveloped viruses, as well as the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by regulated ESCRT-III assembly at the cytoplasmic surface of the membrane to promote the formation and scission of internal vesicles. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission has remained uncharacterized. We show that overexpression of Bro1, or its N-terminal Bro1 domain that binds Snf7, enhances the stability of ESCRT-III. Bro1 binding to the Snf7 subunit of ESCRT-III additionally inhibits Vps4-mediated disassembly of ESCRT-III complexes in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the budding of intralumenal vesicles within endosomes, and the appearance of vesicle budding profiles, vesicles that have not yet separated from the limiting endosomal membrane, as observed by electron microscopy and 3-D electron tomography. These results implicate Bro1 as a regulator of ESCRT-III assembly state, and additionally suggest that Vps4-mediated disassembly of the ESCRT-III complex is important for vesicle membrane scission. We also observed that deletion of ESCRT-III proteins Snf7 or Vps24, or overexpression of Bro1 or the Bro1 domain causes the accumulation of budded yeast cells by flow cytometry. While these proteins have been shown to participate in cell abscission in mammalian cells, a role for these proteins in cell division has not been established in yeast. Our findings suggest that ESCRT-III and Bro1 participate in the abscission step of cytokinesis in yeast, implicating them as conserved effectors of membrane scission.
Wemmer, Megan Anne, "Regulation of ESCRT-III Assembly and Membrane Scission Activity in the Budding Yeast Saccharomyces cerevisiae" (2011). Molecular, Cellular, and Developmental Biology Graduate Theses & Dissertations. 45.