Pkh1/2-dependent phosphorylation of Vps27 regulates ESCRT-I recruitment to endosomes.
Multivesicular endosomes (MVBs) are major sorting platforms for membrane proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydrolase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and ILV formation are achieved by the concerted function of endosomal sorting complex required for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/Phk2 kinases, two yeast orthologues of the 3-phosphoinositide-dependent kinase, phosphorylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine 613 and demonstrate that this phosphorylation is required for proper Vps27 function. Indeed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27(S613A), MVB sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the Vps28-green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT complex recruitment at the endosomal membrane.
Department of Molecular and Cellular Genetics, Unité Mixte de Recherche 7156, Université de Strasbourg and Centre National de la Recherche Scientifique, 67084 Strasbourg, France. morvan@igbmc.fr
Joëlle
J
Morvan
Bruno
B
Rinaldi
Sylvie
S
Friant
D064413:3-Phosphoinositide-Dependent Protein Kinases; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057146:Multivesicular Bodies / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C119627:VPS27 protein, S cerevisiae; D049452:Green Fluorescent Proteins; D010768:Phosphoserine; D064413:3-Phosphoinositide-Dependent Protein Kinases; C118385:PKH1 protein, S cerevisiae; C118386:PKH2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases