Mvb12 Is a Novel Member of ESCRT-I Involved in Cargo Selection by the Multivesicular Body Pathway

Abstract

The multivesicular body (MVB) sorting pathway impacts a variety of cellular functions in eukaryotic cells. Perhaps the best understood role for the MVB pathway is the degradation of transmembrane proteins within the lysosome. Regulation of cargo selection by this pathway is critically important for normal cell physiology, and recent advances in our understanding of this process have highlighted the endosomal sorting complexes required for transport (ESCRTs) as pivotal players in this reaction. To better understand the mechanisms of cargo selection during MVB sorting, we performed a genetic screen to identify novel factors required for cargo-specific selection by this pathway and identified the Mvb12 protein. Loss of Mvb12 function results in differential defects in the selection of MVB cargoes. A variety of analyses indicate that Mvb12 is a stable member of ESCRT-I, a heterologous complex involved in cargo selection by the MVB pathway. Phenotypes displayed upon loss of Mvb12 are distinct from those displayed by the previously described ESCRT-I subunits (vacuolar protein sorting 23, -28, and -37), suggesting a distinct function than these core subunits. These data support a model in which Mvb12 impacts the selection of MVB cargoes by modulating the cargo recognition capabilities of ESCRT-I. INTRODUCTIONThe endosomal system coordinates protein trafficking between various subcellular compartments, including the Golgi, plasma membrane, and lysosome. Cell surface proteins, including activated receptors, that have undergone endocytosis are typically recycled to the plasma membrane or targeted deeper into the endosomal pathway for degradation within the lysosome. Endosomal membrane proteins destined for the lumen of the lysosome undergo an additional sorting reaction during their inclusion into the multivesicular body (MVB) pathway (for review, see Gruenberg and Stenmark, 2004;Babst, 2005). MVBs form when the limiting membrane of the late endosome invaginates and buds into the lumen of the organelle, actively selecting a subset of the proteins from the limiting membrane in the process (Gorden et al., 1978;Haigler et al., 1979). The intralumenal vesicles within the MVB and their contents are exposed to hydrolases within the lysosome as a consequence of heterotypic fusion of these organelles. By contrast, proteins within the limiting membrane of the MVB are delivered to the limiting membrane of the lysosome/vacuole after heterotypic fusion (Odorizzi et al., 1998).Sorting of cargo into the MVB pathway and heterotypic fusion with the lysosme/vacuole leads to delivery into the hydrolytic lumen of the organelle, and therefore entry into this pathway must be highly regulated. Ubiquitination is the best-characterized cis-acting signal mediating entry into the MVB pathway (for reviews, see Katzmann et al., 2002;Hicke and Dunn, 2003;Raiborg et al., 2003). Studies in organisms ranging from the yeast Saccharomyces cerevisiae, to mammalian cells have demonstrated that ubiquitin modification of a variety of MVB cargoes is a requisit...

Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

“…This cluster, which contains 18 genes that encode all 13 nonredundant V-ATPase subunits, 2 overlapping ORFs, and 3 dedicated V-ATPase assembly factors, lacks only VPH1 and STV1, which encode two isoforms of the 100-kDa stator subunit with partially overlapping functions. The secondlargest cluster contains 13 genes corresponding to all known components of ESCRT-I, -II, and -III complexes apart from the newly described ESCRT-I subunit Mvb12 (Chu et al, 2006;Curtiss et al, 2007;Kostelansky et al, 2007;Oestreich et al, 2007) and also includes the ESCRT-associated factors Vps27p, Vps4p, and Bro1p (Bowers and Stevens, 2005). The fact that all three ESCRT subcomplexes were found in a single cluster is consistent with their highly interdependent function in MVB formation.…”

Biogenesis and Function of Multivesicular Bodies

“…Interestingly, the GLUE domain of mammalian Vps36 extends below the flexible C terminus of Ub, where it may be influenced by the context of the isopeptide bond, which may cause a bias for binding particular Ubcargo (Figure 3). Also, deletion of the ESCRT-I subunit Mvb12 causes a selective sorting defect of some Ub-cargoes but not others (Curtiss et al 2007, Oestreich et al 2007b. The function of the ESCRT-I/II supercomplex may be both to promote the release of Ub-cargo from peripheral Ub receptors and to help position Ub-cargo for eventual incorporation into MVBs.…”

The role of ubiquitylation in receptor endocytosis and endosomal sorting

“…ESCRT-0, which consists of HRS [hepatocyte growth factor-regulated tyrosine kinase substrate, also known as HGS and, in yeast, vacuolar protein sorting (Vps) 27p] and STAM (signal-transducing adaptor molecule; Hse1p in yeast), is recruited to endosomal membranes through the interaction of the HRS FYVE domain with phosphatidylinositol 3-phosphate [PtdIns3P] (Burd and Emr, 1998;Gaullier et al, 1998;Raiborg et al, 2001b). ESCRT-0 has the ability to recruit the ESCRT-I complex [which is made up of TSG101 (tumor susceptibility gene 101; Vps23p in yeast), VPS28, VPS37 and MVB12 (multivesicular body subunit 12, also known as FAM125A) (Chu et al, 2006;Katzmann et al, 2001;Kostelansky et al, 2007;Morita et al, 2007;Oestreich et al, 2007;Stuchell et al, 2004)] through an interaction between HRS and TSG101 (Bache et al, 2003;Katzmann et al, 2003;Lu et al, 2003). ESCRT-I and ESCRT-II, which comprise a single VPS36 (also known as EAP45), a single VPS22 and two VPS25 subunits, can form a supercomplex through interactions between VPS28 and VPS36 (also see below) (Gill et al, 2007;Im and Hurley, 2008 Urbe et al, 2003).…”