Sna3 Is an Rsp5 Adaptor Protein that Relies on Ubiquitination for Its MVB Sorting

Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, 52246, USA.
Traffic (Impact Factor: 4.35). 12/2011; 13(4). DOI: 10.1111/j.1600-0854.2011.01326.x
Source: PubMed


The process in which ubiquitin (Ub) conjugation is required for trafficking of integral membrane proteins into multivesicular bodies (MVBs) and eventual degradation in the lumen of lysosomes/vacuoles is well defined. However, Ub-independent pathways into MVBs are less understood. To better understand this process, we have further characterized the membrane protein Sna3, the prototypical Ub-independent cargo protein sorted through the MVB pathway in yeast. We show that Sna3 trafficking to the vacuole is critically dependent on Rsp5 ligase activity and ubiquitination. We find Sna3 undergoes Ub-dependent MVB sorting by either becoming ubiquitinated itself or associating with other ubiquitinated membrane protein substrates. In addition, our functional studies support a role for Sna3 as an adaptor protein that recruits Rsp5 to cargo such as the methionine transporter Mup1, resulting in efficient Mup1 delivery to the vacuole.

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    • "Rsp5, the sole member of the Nedd4 E3 ligase family in S. cerevisiae, has been implicated in various steps in intracellular trafficking, including endocytosis of plasma membrane proteins [3] such as the Fur4 uracil permease [4] and the Mat α receptor Ste2 [5]. Apart from a key initial role in substrate ubiquitination immediately preceding target internalization, Rsp5 is involved in other steps in vesicle trafficking, such as sorting at the multivesicular body (MVB) [6], [7], [8] that precedes cargo delivery and degradation at the vacuole, and more recently, in mediating Golgi to ER trafficking [9]. "
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    ABSTRACT: The yeast HECT-family E3 ubiquitin ligase Rsp5 has been implicated in diverse cell functions. Previously, we and others [1], [2] reported the physical and functional interaction of Rsp5 with the deubiquitinating enzyme Ubp2, and the ubiquitin associated (UBA) domain-containing cofactor Rup1. To investigate the mechanism and significance of the Rsp5-Rup1-Ubp2 complex, we examined Rsp5 ubiquitination status in the presence or absence of these cofactors. We found that, similar to its mammalian homologues, Rsp5 is auto-ubiquitinated in vivo. Association with a substrate or Rup1 increased Rsp5 self-ubiquitination, whereas Ubp2 efficiently deubiquitinates Rsp5 in vivo and in vitro. The data reported here imply an auto-modulatory mechanism of Rsp5 regulation common to other E3 ligases.
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    • "As confirmation, we made homozygous hse1D diploids from SF838-9D and SEY6210, in which the BRO1 gene was knocked out of either haplotype. The hse1D diploids having only the Y359 Bro1 from SF838-9D showed a strong MVB sorting defect as assessed by the localization of Sna3-GFP (Figure 1C), a well-characterized MVB cargo (Macdonald et al., 2012b). In contrast, hse1D homozygotes with the SEY6210 allele of BRO1 (C359) sorted Sna3-GFP normally. "
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    ABSTRACT: Sorting of ubiquitinated membrane proteins into lumenal vesicles of multivesicular bodies is mediated by the Endosomal Sorting Complex Required for Transport (ESCRT) apparatus and accessory proteins such as Bro1, which recruits the deubiquitinating enzyme Doa4 to remove ubiquitin from cargo. Here we propose that Bro1 works as a receptor for the selective sorting of ubiquitinated cargos. We found synthetic genetic interactions between BRO1 and ESCRT-0, suggesting that Bro1 functions similarly to ESCRT-0. Multiple structural approaches demonstrated that Bro1 binds ubiquitin via the N-terminal trihelical arm of its middle V domain. Mutants of Bro1 that lack the ability to bind Ub were dramatically impaired in their ability to sort Ub-cargo membrane proteins, but only when combined with hypomorphic alleles of ESCRT-0. These data suggest that Bro1 and other Bro1 family members function in parallel with ESCRT-0 to recognize and sort Ub-cargos.
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    ABSTRACT: Cellular responsiveness to many neuromodulators is controlled by endocytosis of the transmembrane receptors that transduce their effects. Endocytic membrane trafficking of particular neuromodulator receptors exhibits remarkable diversity and specificity, determined largely by molecular sorting operations that guide receptors at trafficking branchpoints after endocytosis. In this Review, we discuss recent progress in elucidating mechanisms mediating the molecular sorting of neuromodulator receptors in the endocytic pathway. There is emerging evidence that endocytic trafficking of neuromodulator receptors, in addition to influencing longer-term cellular responsiveness under conditions of prolonged or repeated activation, may also affect the acute response. Physiological and pathological consequences of defined receptor trafficking events are only now being elucidated, but it is already apparent that endocytosis of neuromodulator receptors has a significant impact on the actions of therapeutic drugs. The present data also suggest, conversely, that mechanisms of receptor endocytosis and molecular sorting may themselves represent promising targets for therapeutic manipulation.
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