title: 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.
abstract: Human Golgi-localized, γ-ear-containing, ADP-ribosylation factor-binding proteins (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 Vps27, Hrs, Stam (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 autoinhibitory 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-GGA and TOM1 (GAT) and glutathione S-transferase-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 wild-type 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 trans-Golgi network (TGN)-prevacuolar compartment (PVC) transport. Thus yeast Gga adaptors facilitate TGN-PVC transport by direct binding of noncanonical phosphoregulated Gga-binding sites in cargo molecules.
authors: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors: Mithu
authors: M
authors: De
authors: Mohamed E
authors: ME
authors: Abazeed
authors: Robert S
authors: RS
authors: Fuller
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D011992:Endosomes / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D010766:Phosphorylation; D043484:Proprotein Convertases / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014617:Vacuoles / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C497361:Gga2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae