Signaling by K63-linked polyubiquitin in autophagy-related vesicular trafficking

Scavone, Francesco, author
Cohen, Robert, advisor
Yao, Tingting, advisor
Di Pietro, Santiago, committee member
Ordway, Diane, committee member
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In eukaryotic cells, endocytosis and autophagy represent fundamental pathways that contribute to cell homeostasis. Intersection of the endocytic and autophagosomal trafficking routes has been previously recognized, as both systems share specific compartments contributing to cargo degradation via the lysosome, and both use an identical subset effectors involved in vesicular trafficking and membrane fusion. By helping to target proteins to the lysosome, ubiquitin (Ub) also acts as a common factor in both pathways. The relative contributions of endocytic and autophagy pathways to degradation of ubiquitinated cargo are mostly unknown. A critical unanswered question is how ubiquitinated cargo is targeted to a particular degradative pathway. Ub can be conjugated to other proteins as a single Ub or chains of multiple Ub (polyUb). Many types of polyUb, distinguished by different Ub–Ub linkages, are present in cells, where they are thought to be recognized by decoders (i.e., receptors or adaptors) to signal various biological processes. K63-polyUb has been implicated in endocytosis of cell surface receptors, budding of viral proteins, and autophagy of damaged organelles and invading pathogens. However, the extent and details of the relationship between K63-polyUb and lysosomal targeting via either endocytosis or autophagy are poorly understood. These deficiencies are due in part to the lack of tools to study Ub-dependent signaling. We have previously developed a genetically-encoded specific K63-polyUb sensor, "Vx3," to facilitate identification, localization, and functional characterization of signaling by K63-polyUb in vivo [Sims et al., Nat. Methods (2012)]. When Vx3 is expressed in cells, it localizes to cytoplasmic foci that colocalize with the autophagic transmembrane protein ATG9A, the autophagic cargo receptor p62 (SQSTM1), which preferentially bind K63-polyUb, and late endosomal/lysosomal markers. Proteins identified by mass spectrometry and likely modified by K63-polyUb appear to be transmembrane proteins known to recycle between endosomes and plasma membrane, such as transferrin receptor (TfR) and major histocompatibility complex I (MHC-I). Here, we show that K63-polyUb modifies endocytic transmembrane proteins to signal their delivery to the lysosomes. Fluorescence and electron microscopy data revealed that Vx3 accumulates K63-polyUb-decorated vesicles into cytoplasmic foci at an intermediate stage of the transmembrane protein degradation pathway; these foci include components of both endocytic and autophagy membranes. We observed that K63-polyUb-decorated vesicles co-clustering with ATG9A are found juxtaposed to endolysosomes, consistent with the idea of fusion block imposed by Vx3. By using a system to inducibly disable Vx3 inhibition, we tracked delivery of K63-polyubiquitinated cargo to the highly acidic and degradative lysosomal compartments. We also report that ESCRT dysfunction leads to accumulation Vx3 foci, indicating an augmentation of K63-polyubiquinated cargo destined to lysosomal degradation via the multivesicular late endosomes. Despite the established role for endocytosis in cargo sorting and recycling, there is evidence that autophagy may also play a role in regulating these events. By using Vx3 to inhibit autophagy-related membrane trafficking, our study also reveal a functional crosstalk between the Ub-dependent sorting of endocytic cargo into multivesicular late endosomes and the retromer-mediated retrieval of transmembrane proteins from maturing endosomes.
2019 Fall.
Includes bibliographical references.
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