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Probing unconventional vesicular trafficking with K63-polyubiquitin sensors

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dc.contributor.authorLian, Sharon, author
dc.contributor.authorCohen, Robert, advisor
dc.contributor.authorYao, Tingting, advisor
dc.contributor.authorReist, Noreen, committee member
dc.date.accessioned2019-09-10T14:36:04Z
dc.date.available2020-09-03T14:36:28Z
dc.date.issued2019
dc.description.abstractFor 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.
dc.format.mediumborn digital
dc.format.mediummasters theses
dc.identifierLian_colostate_0053N_15588.pdf
dc.identifier.urihttps://hdl.handle.net/10217/197360
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartof2000-2019
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.
dc.subjectGolgi bypass
dc.subjecttransmembrane proteins
dc.subjectK63-linked ubiquitin
dc.subjectER-endolysosome
dc.titleProbing unconventional vesicular trafficking with K63-polyubiquitin sensors
dc.typeText
dcterms.embargo.expires2020-09-03
dcterms.embargo.terms2020-09-03
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineBiochemistry and Molecular Biology
thesis.degree.grantorColorado State University
thesis.degree.levelMasters
thesis.degree.nameMaster of Science (M.S.)

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