Recruitment of the ESCRT machinery to a putative seven-transmembrane-domain receptor is mediated by an arrestin-related protein.
Mammalian arrestins have a major role in the intracellular trafficking of seven-transmembrane (7TM) receptors. The fungal ambient pH signaling pathway involves an arrestin-related protein, PalF/Rim8, and the ESCRT (endosomal sorting complex required for transport) machinery. We found that in Saccharomyces cerevisiae, Rim8 binds to both the putative 7TM pH sensor Rim21 and the ESCRT-I subunit Vps23. We show that an SXP motif in Rim8 mediates binding to the Vps23 ubiquitin E2 variant (UEV) domain and that a monoubiquitinated residue near the SXP motif contributes to this interaction. We present evidence that Rim8 ubiquitination is dependent on the Rsp5 E3 ubiquitin ligase and triggered upon binding of Vps23 UEV to both the SXP motif and ubiquitin, thus suggesting a two-step binding mechanism. We further show that Rim8 coimmunoprecipitates with ESCRT-I subunits Vps23 and Vps28, supporting the idea that binding of Rim8 to Vps23 mediates the association of Rim8 with the ESCRT-I complex. Fluorescence microscopic analyses indicate that overexpressed Rim8 and Vps23 colocalize at cortical punctate structures, providing additional evidence of the interaction between these two proteins. Strikingly, our findings indicate that evolutionary conserved mechanisms control the recruitment of the ESCRT machinery to Pal/Rim proteins in fungi and retroviral Gag proteins in animal cells.
Herrador
Antonio
A
Instituto de Investigaciones Biomédicas CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain.
Herranz
Silvia
S
Lara
David
D
Vincent
Olivier
O
D020816:Amino Acid Motifs; D019393:Arrestin / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D054875:Ubiquitination
D019393:Arrestin; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D047908:Intracellular Signaling Peptides and Proteins; D021122:Protein Subunits; D011956:Receptors, Cell Surface; C547025:Rim21 protein, S cerevisiae; C547024:Rim8 protein, S cerevisiae; C425333:STP22 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C102954:VPS28 protein, S cerevisiae