A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination

Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52246, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 01/2011; 192(2):229-42. DOI: 10.1083/jcb.201008121
Source: PubMed


ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination.

11 Reads
    • "We therefore examined whether ubiquitination plays a role in the NVT pathway by using a DUb (deubiquitinating enzyme) fusion approach (Stringer and Piper, 2011). Similar to the observations in S. cerevisiae (Stringer and Piper, 2011), fusing a DUb, but not its enzymatically inactive form, to MVB pathway cargos such as Ncs2 prevented the transport into vacuoles (Figure S4H). When Nbr1 was fused to either one of two DUbs, its vacuolar targeting was abolished, whereas Nbr1 fused to the catalytically inactive forms of these DUbs still accumulated inside vacuoles (Figures 4E and 4F). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Autophagy transports cytosolic materials into lysosomes/vacuoles either in bulk or selectively. Selective autophagy requires cargo receptor proteins, which usually link cargos to the macroautophagy machinery composed of core autophagy-related (Atg) proteins. Here, we show that fission yeast Nbr1, a homolog of mammalian autophagy receptor NBR1, interacts with and facilitates the transport of two cytosolic hydrolases into vacuoles, in a way reminiscent of the budding yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy. We term this pathway Nbr1-mediated vacuolar targeting (NVT). Surprisingly, unlike the Cvt pathway, the NVT pathway does not require core Atg proteins. Instead, it depends on the endosomal sorting complexes required for transport (ESCRTs). NVT components colocalize with ESCRTs at multivesicular bodies (MVBs) and rely on ubiquitination for their transport. Our findings demonstrate the ability of ESCRTs to mediate highly selective autophagy of soluble cargos, and suggest an unexpected mechanistic versatility of autophagy receptors.
    Molecular cell 09/2015; 59(6). DOI:10.1016/j.molcel.2015.07.034 · 14.02 Impact Factor
  • Source
    • "ILV formation and cargo sorting are mediated by the endosomal sorting complex required for transport (ESCRT) machinery (Raiborg and Stenmark, 2009). Previous studies indicate that K63-linked polyubiquitylation of cargo proteins specifically functions as an ESCRT-dependent sorting signal to ILVs rather than as an internalization signal (Huang et al., 2006; Barriere et al., 2007; Lauwers et al., 2010; Erpapazoglou et al., 2012), although other reports suggest that multiple or even single monoubiquitylation is sufficient for MVB sorting (Stringer and Piper, 2011). Thus, the exact role of K63- linked ubiquitylation in endocytic processes remains controversial. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In Caenorhabditis elegans, fertilization triggers endocytosis and rapid turnover of maternal surface membrane proteins in lysosomes, although the precise mechanism of this inducible endocytosis is unknown. We found that high levels of K63-linked ubiquitin chains transiently accumulated on endosomes upon fertilization. Endocytosis and the endosomal accumulation of ubiquitin were both regulated downstream of the anaphase-promoting complex, which drives the oocyte's meiotic cell cycle after fertilization. The clearance of maternal membrane proteins and the accumulation of K63-linked ubiquitin on endosomes depended on UBC-13 and UEV-1, which function as an E2 complex that specifically mediates chain elongation of K63-linked polyubiquitin. CAV-1-GFP, an endocytic cargo protein, was modified with K63-linked polyubiquitin in a UBC-13/UEV-1-dependent manner. In ubc-13 or uev-1 mutants, CAV-1-GFP and other membrane proteins were internalized from the plasma membrane normally after fertilization. However, they were not efficiently targeted to the multivesicular body (MVB) pathway but recycled to the cell surface. Our results suggest that UBC-13-dependent K63-linked ubiquitylation is required for proper MVB sorting rather than for internalization. These results also demonstrate a developmentally controlled function of K63-linked ubiquitylation.
    Development 03/2014; 141(6):1324-31. DOI:10.1242/dev.103044 · 6.46 Impact Factor
  • Source
    • "pPL5025 pPL5026 chimeric Bro1-2xHA with V domain from pPL4781 pRS315, pRS316 pPL3797 pPL4069 S.castelli Bro1 V (370-410-SGD-529-646) in pET151 Mup1-GFP in pRS315 Linton Traub Yeast clathrin (1-163) NTD in pET28a S.castelli Bro1 V I377A E380R L386A in pET151 pPL5074 pPL967 Mouse Cks1 in pET151 pPL5056 S.castelli Bro1 V E373C in pET151 pPL5057 S.castelli Bro1 V L386A in pET151 pPL5060 S.castelli Bro1 V I377A E380R in pET151 pPL5061 S.castelli Bro1 V E389A M390A E391A in pET151 LEU2 and URA3 yeast expression plasmids S.castelli Bro1 V R387E K388E in pET151 Sna3-GFP in pRS315 CUP1 promoter Gap1-GFP in pRS315 Ste3-GFP in pRS315 pPL4945 pPL4946 pPL4948 pPL4950 pPL4951 pPL4953 pPL4959 pPL4955 pPL4957 S.castelli Bro1 V R564D K567D in pET151 S.castelli Bro1 V L566A K567A E568A in pET151 S.castelli Bro1 V K567A E568A E569A in pET151 S.castelli Bro1 V R572A T573A M574A in pET151 S.castelli Bro1 V T617A T618A R619A in pET151 S.castelli Bro1 V L605A F606A E607A in pET151 S.castelli Bro1 V L610A K612E in pET151 pPL4469 pPL2089 pPL3791 This study pPL3878 GFP-Ub in pRS315 pPL4148 CUP1 promoter HIS3-UL36-3xHA pPL5079 pPL5080 chimeric Bro1-2xHA with V domain from pPL4445 pPL5099 CUP1 promoter Bro1-UL36-3xHA in pRS316 CUP1 promoter Bro1-Ubp7-3xHA in pRS316 CUP1 promoter BroDomain-UL36-3xHA in pRS316 pPL5034 pPL5059 chimeric Bro1-2xHA with V domain from pPL4981 pPL4178 CUP1promoter Bro1-HA from S.cerevisiae CUP1 promoter MIT-UL36-3xHA in pRS316 pPL5113 pPL5073 Bovine clathrin (1-163) NTD-NEMO in pET28a Relevant to Figures 1, 2 and 7 MATα leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 lys2-801 suc2-Δ9 mel This Study Scott Moye-Rowley, U of Iowa PLY3091 SEY6210(EE10) MATa hse1∆::TRP1 This Study PLY4249 SEY6210(EE10) MATa hse1∆::TRP1 bro1∆::URA3 This Study PLY4259 SF838-9D; PEP4 hse1D::KanR bro1∆::URA3 This Study Bilodeau et al., 2003 Stringer and Piper, 2011 "
    Dataset: mmc1
Show more

Preview (2 Sources)

11 Reads
Available from