Probing unconventional vesicular trafficking with K63-polyubiquitin sensors
Date
2019
Authors
Lian, Sharon, author
Cohen, Robert, advisor
Yao, Tingting, advisor
Reist, Noreen, committee member
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Abstract
For signaling purposes, the small protein ubiquitin (Ub) acts as a post-translational modification. Ub can polymerize with diverse Ub-Ub chain linkages which are involved in numerous cellular mechanisms. To investigate processes mediated by a particular Ub linkage, tools selective against specific forms of polyUb are useful. Vx3 is a previously developed sensor that specifically binds K63-linked polyUb with high affinity and acts as a competitive inhibitor by blocking K63-polyUb-dependent signaling. When expressed in cells, Vx3 forms stable cytoplasmic foci that co-localize with autophagy related protein 9A (ATG9A) and late endosomal/lysosomal markers. However, Vx3 foci only co-localize with the autophagy marker LC3 upon selective autophagy induction. Proteins associated with Vx3 were identified through Vx3 co-immunoprecipitation and mass spectrometry analysis. The most abundant were plasma membrane proteins including transferrin receptor (TfR) and major histocompatibility complex I (MHC-I), which co-localized into cytoplasmic foci with Vx3. Biochemical and confocal microscopy analyses revealed that TfR at Vx3 foci is K63-polyubiquitinated, originated from the ER, and bypassed the Golgi apparatus via a non-canonical trafficking pathway. In addition, Vx3 was modified to allow inducible release of bound K63-polyUb. By disabling Vx3, tracking of the ubiquitinated proteins to observe their downstream activities becomes possible. This thesis preliminarily identifies K63-polyUb as a signal for what is possibly a quality control pathway and offers tools for further investigation in context of Vx3.
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Subject
Golgi bypass
transmembrane proteins
K63-linked ubiquitin
ER-endolysosome