A Membrane Coat Complex Essential for Endosome-to-Golgi Retrograde Transport in Yeast

Abstract

We have recently characterized three yeast gene products (Vps35p, Vps29p, and Vps30p) as candidate components of the sorting machinery required for the endosome-to-Golgi retrieval of the vacuolar protein sorting receptor Vps10p (Seaman, M.N.J., E.G. Marcusson, J.-L. Cereghino, and S.D. Emr. 1997. J. Cell Biol. 137:79–92). By genetic and biochemical means we now show that Vps35p and Vps29p interact and form part of a multimeric membrane-associated complex that also contains Vps26p, Vps17p, and Vps5p. This complex, designated here as the retromer complex, assembles from two distinct subcomplexes comprising (a) Vps35p, Vps29p, and Vps26p; and (b) Vps5p and Vps17p. Density gradient fractionation of Golgi/endosomal/vesicular membranes reveals that Vps35p cofractionates with Vps5p/Vps17p in a vesicle-enriched dense membrane fraction. Furthermore, gel filtration analysis indicates that Vps35p and Vps5p are present on a population of vesicles and tubules slightly larger than COPI/coatomer-coated vesicles. We also show by immunogold EM that Vps5p is localized to discrete regions at the rims of the prevacuolar endosome where vesicles appear to be budding. Size fractionation of cytosolic and recombinant Vps5p reveals that Vps5p can self-assemble in vitro, suggesting that Vps5p may provide the mechanical impetus to drive vesicle formation. Based on these findings we propose a model in which Vps35p/Vps29p/Vps26p function to select cargo for retrieval, and Vps5p/Vps17p assemble onto the membrane to promote vesicle formation. Conservation of the yeast retromer complex components in higher eukaryotes suggests an important general role for this complex in endosome-to-Golgi retrieval.

Characterization of anERADGene asVPS30/ATG6Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble Z Variant of Human α-1 Proteinase Inhibitor (A1PiZ) and Another for Aggregates of A1PiZ

“…The data presented above indicate a requirement for both VPS30 and VPS38 during A1PiZ degradation, and Vps30p and Vps38p are necessary for the recycling of Vps10p, a sorting receptor that plays a role in trafficking CPY (Marcusson et al, 1994;Seaman et al, 1997Seaman et al, , 1998Burda et al, 2002), proteinase A, aminopeptidase Y (Jorgensen et al, 1999), and some misfolded proteins from the Golgi to the vacuole (Hong et al, 1996;Holkeri and Makarow, 1998). We therefore asked whether VPS10 was also required for A1PiZ degradation.…”

Early Stages of the Secretory Pathway, but Not Endosomes, Are Required for Cvt Vesicle and Autophagosome Assembly inSaccharomyces cerevisiae

“…We examined the localization of GFP-Ape1 in the class E mutants to verify that maturation correlated with vacuolar delivery. GFP-Ape1 was localized to the vacuolar lumen, not the class E compartment, in the class E vps mutants, indicating that normal transport had taken place (our unpublished data).The retromer complex is an essential part of the sorting machinery required for retrieval from the late endosome to the Golgi and is composed of the VPS35, VPS29, VPS26, VPS17, and VPS5 gene products, which fall into several of the VPS classes (Seaman et al, 1998). Similar to the class E mutants, defects in these proteins cause significant blocks in transport processes that depend on the late endosome.…”

“…This organelle corresponds to the enlarged late endosome typically accumulated in all class E vps deletants (Vida and Emr, 1995;Rieder et al, 1996;Babst et al, 1997). In retromer mutants, vacuolar proteases such as Prc1 (CPY) and Pep4 are partially missorted to the periplasmic space in the precursor form (Seaman et al, 1997;Seaman et al, 1998).To verify the phenotype of the vps5⌬, vps29⌬, and vps35⌬ strains, the same cell extracts analyzed in Figure 1B were probed with antisera recognizing these two hydrolases. As shown in Figure 1D, unprocessed Prc1 (p2 or Golgi-modified form) and Pep4 (prPep4) were accumulated in retromer deletants but not in wild-type or atg1⌬ cells, confirming that the former displayed the expected phenotypes of vps mutants.…”

Tri sélectif et recyclage

“…Ce complexe fonctionne dans le transport rétrograde de protéines et (peut-être) de lipides vers le réseau TGN. Ainsi, chez la levure, le retromer promeut le recyclage de Vps10p, le transporteur des hydrolases vacuolaires solubles, depuis les endosomes prévacuolaires jusqu'au TGN [17]. Ce transport rétrograde permet de réutiliser le récepteur de tri Vps10 pour plusieurs cycles de transport des hydrolases vacuolaires.…”

“…Ces articles démon-trent par ailleurs que Wntless est un récepteur de tri intracellulaire. Le retromer a été initialement identifié chez la levure comme étant requis pour le routage intracellulaire des hydrolases vacuolaires [17]. Chez le nématode, la perte de fonction du retromer induit des phénotypes restreints observables dans la partie postérieure de l'animal, et similaires à ceux qu'entraîne une altération de la voie de signalisation WNT [14,19].…”