A Family of Tetraspans Organizes Cargo for Sorting into Multivesicular Bodies

Abstract

SUMMARY The abundance of cell surface membrane proteins is regulated by internalization and delivery into intralumenal vesicles (ILVs) of multivesicular bodies (MVB). Many cargoes are ubiquitinated, allowing access to an ESCRT-dependent pathway into MVBs. Yet, how non-ubiquitinated proteins, such as Glycosylphosphatidylinisotol-anchored proteins, enter MVBs is unclear, supporting the possibility of mechanistically distinct ILV biogenesis pathways. Here we show a family of highly ubiquitinated tetraspan Cos proteins provide a Ub-signal in trans, allowing sorting of non-ubiquitinated MVB cargo into the canonical ESCRT- and Ub-dependent pathway. Cos proteins create discrete endosomal subdomains that concentrate Ub-cargo prior to their envelopment into ILVs and the activity of Cos proteins is required not only for efficient sorting of canonical Ub-cargo but is also essential for sorting non-ubiquitinated cargo into MVBs. Expression of these proteins increases during nutrient stress though a NAD+/Sir2-dpendent mechanism that in turn accelerates the down-regulation of a broad range of cell surface proteins.

Cell surface recycling in yeast: mechanisms and machineries

“…As discussed above, one of the problems in studying the trafficking of most cell surface proteins is that they ultimately become modified by Ub and are sent into the MVB pathway. This problem even plagues GFP-Snc1, which is sent to the vacuole when cells are simply grown past log phase, [8] and Figure 2. Recently, we developed a tool that negates the effects of ubiquitination on cargoes: one such example is a chimeric protein based on the recycling cargo Ste3 fused to catalytic domain of a DUb.…”

“…One is the multivesicular body (MVB) pathway, which packages protein cargo into intralumenal vesicles that form from the limiting membrane of endosomes and that are ultimately delivered to the lysosome for degradation or to the PM for secretion as exosomes. The canonical signal for entry into the MVB pathway is ubiquitin (Ub), which can be attached directly to cargo as a sorting signal [7] or attached to a protein associated with cargo to allow sorting in trans [8-10]. Alternatively, cargo can follow a ‘retrograde’ route, taking them back to the TGN where they re-join the secretory pathway [11, 12].…”

“…Similarly, the sugar transporter Jen1 can transit back to the PM from endosomes once the signal that drives its Rod1-Rsp5 mediated ubiquitination is attenuated [25]. In addition, disruption of the large polymers of ESCRT-III that might trap ubiquitinated proteins on endosomes [26] or deletion of a newly described yeast family of cargo carrier proteins that sequester ubiquitinated proteins on endosomes [8], increases levels of cell surface proteins that are otherwise efficiently sent to the MVB pathway. In agreement with these findings, engineering yeast to deubiquitinate cargo once it contacts the ESCRT sorting machinery leads to high steady state levels of a variety of cell surface proteins, supporting the idea that deubiquitination can reveal a ‘default’ pathway for many cell surface proteins - not just cargo with specific sorting signals for retromer - to regain access to the PM after release from the clutches of the Ub-mediated degradation pathway [27].…”

“…Here, the yeast methionine permease (Mup1), was sent to late endosomes by inducing its Art1-Rsp5-dependent ubiquitination, and was then subjected to chemically induced deubiquitnation using the FKBP-FRB system to attach a deubiquitinating enzyme directly to the cargo. While other ubiquitinated cargo remained in endosomes, the Mup1~DUb complex was transported back to the PM where it remained stable [8]. Importantly, Mup1 is not known to have specialized signals for entry into the known endosomal retrieval pathways.…”

Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgarisSuf1 transporter

“…Besides faster growth, the mutations may also be responsible for lowering the residual sucrose concentration in the accelerostat reactor (from 8 to 2.5 g/L; see Section 3). Chromosomal duplications were also found in the genome of the evolved strains (Table , Figure S2), affecting genes that encode proteins involved in transporter sorting, ubiquitination, and degradation: COS10 , SEC12 , and SIS1 , which are present on chromosome 14 (Luke, Sutton, & Arndt, ; Macdonald et al, ; Nakano, Brada, & Schekman, ); CUR1 and SEC23 from chromosome 16 (Alberti, ) and UBC7 found on chromosome 13 (Hiller, Finger, Schweiger, & Wolf, ). However, these genomic alterations are unlikely to be the main cause of the improved specific growth rate of the evolved strains because reverse engineering of a mutated version of PvSUF1 in unevolved S. cerevisiae sufficed to generate a strain that grew as fast as the fastest‐growing evolved strain (Table ).…”