De M, et al. (2012) Direct Binding of the Kex2p Cytosolic Tail to the VHS Domain of Yeast Gga2p Facilitates TGN to Prevacuolar Compartment Transport and is Regulated by Phosphorylation. Mol Biol Cell ()
Abstract: Human GGAs bind directly to acidic dileucine sorting motifs in the cytosolic tails (C-tails) of intracellular receptors. Despite evidence for a role in recruiting ubiquitinated cargo, it remains unclear whether yeast Ggas also function by binding peptide sorting signals directly. Two-hybrid analysis shows that the Gga1p and Gga2p VHS domains both bind a site in the Kex2p C-tail and that the Gga2p VHS domain binds a site in the Vps10p C-tail. Binding requires deletion of an apparently auto-inhibitory sequence in the Gga2p hinge. Ser(780) in the Kex2p C-tail is crucial for binding: an Ala substitution blocks but an Asp substitution permits binding. Biochemical assays using purified Gga2p VHS-GAT and GST-Kex2p C-tail fusions show that Gga2p binds directly to the Kex2p C-tail, with relative affinities: Asp(780)>Ser(780)>Ala(780). Affinity-purified antibody against a peptide containing phospho-Ser-(780) recognizes WT Kex2p but not S(780)A Kex2p, showing that Ser(780) is phosphorylated in vivo; phosphorylation of Ser(780) is up-regulated by cell-wall damaging drugs. Finally, mutation of Ser(780) alters trafficking of Kex2p both in vivo and in cell-free TGN-PVC transport. Thus, yeast Gga adaptors facilitate TGN-PVC transport by direct binding of non-canonical phosphoregulated Gga-binding sites in cargo molecules.
|Status: Epub ahead of print||Type: Journal Article||PubMed ID: 23264466|
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