ORCID

http://orcid.org/0000-0001-8891-4509

Date of Award

Summer 8-15-2020

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Neurosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

ESCRTs (Endosomal Sorting Complex Required for Transport) are a modular set of proteins with membrane remodeling activities that include the formation and release of intralumenal vesicles (ILVs) to generate multivesicular endosomes. ESCRT-III filaments have an established role in membrane fission for ILV formation and the topologically related processes of viral budding and cytokinesis. Among the 12 ESCRT-III proteins most have established roles in ILV formation, but the cellular roles of IST1 remain elusive. We found that IST1 and another ESCRT-III subunit CHMP1B form filaments that spiral around the outside or cytoplasmic surface of membrane tubules. Consistent with a role in tubule-based sorting processes—and a possible role in fission—CHMP1B and IST1 are present in puncta along endosomal tubules and live-cell imaging showed that depleting either one stabilized elongated Rab5 endosomes. We also identified a novel IST1 interacting protein SNX15, which promotes the recruitment of IST1 to a microdomain distinct from those established for cargo degradation and cargo recycling. Proximity biotinylation studies (BioID2) using IST1 and SNX15 established that both proteins associate with a common set of factors involved in endosomal recycling including clathrin and branched actin regulators, while IST1 has additional unique partners that bind uniquely via ESCRT-III and MIT domain interactions. Using live-cell microscopy we found that SNX15 and CHMP1B alternately control IST1 distribution and its relationship to clathrin and branched actin—as well as how IST1 affects the distribution of transferrin-positive recycling tubules within the cell. Using the transferrin receptor (TfnR) as an endosomal cargo, we established that IST1 regulates sorting and trafficking from the early/sorting endosome to the tubular endocytic recycling compartment (ERC) present in the perinuclear region. Loss of IST1 impaired the sorting of TfnR by this pathway and led to its accumulation in abnormal peripheral endosomes that were positive for the endosomal clathrin adaptor AP-1. These results demonstrate that IST1 is a multifunctional adaptor involved in clathrin-based recycling on the endosome either by interacting with clathrin-binding SNX15 or endo-tubule membrane constricting CHMP1B filaments—and possibly functions with other actin-based sorting mechanisms on the endosome.

Language

English (en)

Chair and Committee

Phyllis I. Hanson

Committee Members

Michael L. Nonet, Ken Blumer, Silvia Jansen, David J. Kast,

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