Reference: De M, et al. (2013) 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 24(4):495-509

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Abstract


Human Golgi-localized, gamma-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.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't | Research Support, N.I.H., Extramural
Authors
De M, Abazeed ME, Fuller RS
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