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

Abstract

Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. INTRODUCTIONEndosomal protein sorting is a highly conserved cellular process that is required for receptor down-regulation, viral replication, and development (Katzmann et al., 2002;Morita and Sundquist, 2004). Much progress has been made in recent years in identifying distinct multiprotein complexes that regulate the fusion of primary endocytic vesicles, the maturation of an early endosome into a multivesicular body (MVB), and the recycling of proteins and lipids to other organelles (Gruenberg and Stenmark, 2004;Bonifacino and Rojas, 2006). The ESCRT-I, -II, and -III complexes (endosomal-sorting complex required for transport), which act in sequence to regulate the formation of internal vesicles at the MVB, play a central role in endosome biogenesis (Katzmann et al., 2002;Hurley and Emr, 2006). Other transport complexes regulate the targeting and fusion of endosomes or endosome-derived vesicles with other organelles (Bowers and Stevens, 2005;Bonifacino and Rojas, 2006).Many components of the endosomal-sorting machinery were first identified in yeast genetic screens for mutants that are defective in protein transport to the yeast vacuole, which requires functional endosomal sorting and recycling (Bowers and Stevens, 2005). Most of these components have a conserved role in endosome biogenesis in higher cells. Mammalian homologues have been identified for most if not all yeast ESCRT subunits (von Schwedler et al., 2003;Hurley and Emr, 2006), and at least some of the machinery that regulates recycling from the MVB is also well conserved. For example, the five-subunit retromer complex that mediates endosome-to-trans-Golgi network (TGN) transport in yeast is associated with tubular regions of the endosome in mammalian cells and mediates retrograde transport (Arighi et al., 2004;Seaman, 2004). In the past 20 years, classical genetic screens have identified more than 50 vacuole protein-sorting (VPS) genes (Bowers and Stevens, 2005). Surprisingly, ...

Endospanins Regulate a Postinternalization Step of the Leptin Receptor Endocytic Pathway

“…Because the principles of membrane trafficking are conserved from yeast to neurons, results obtained with HeLa cells are likely to be valid for other cell types. These features are quite similar to those of Vps55p, a member of this family expressed in S. cerevisiae, which is implicated in late endosome-to-vacuole transport (13,26). This suggests that the members of this family provide a function in membrane traffic that is common to phylogenetically distant eukaryotes.…”

“…It functions as a regulator of TGN to apical plasma membrane traffic in the secretory pathway of polarized cells (31). Vps55p appears to be such a general regulator of the membrane traffic between endosomes and the vacuole (13,26). Our results indicate that endospanins function in endosomes.…”

“…On the contrary, their different intracellular localization at steady state rather suggests that both proteins could fulfill related functions in separate protein complexes. In yeast, there is evidence that Vps55p is part of a functional protein complex that also includes Vps68p, another small membrane protein with four putative transmembrane domains (26). Two genes for Vps68p-like proteins are also found in the human genome, 4 suggesting that the whole complex has been duplicated in humans.…”

LEPROT and LEPROTL1 cooperatively decrease hepatic growth hormone action in mice

“…In yeast, Vps55p and hLEPROT are localized in the late endosome and participate in the vacuolar targeting of CPY (15). Vps55p also participates in the downregulation of the pheromone receptor ste3p (16). It has been suggested that LEPROT plays a role in late endocytotic steps (17).…”

“…It is likely that LEPROT family members are part of functional protein complexes that include at least 1 small membrane protein with 4 putative transmembrane domains. Indeed, Vps55p interacts with Vps68p, a small tetraspan protein (16). Recently, it has been demonstrated in vitro that LEPROT negatively regulates leptin receptor cell-surface expression and that in vivo LEPROT silencing in mouse hypothalamic arcuate nucleus increases leptin signaling (17).…”