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

Mithu De, Mohamed E. Abazeed, Robert S. Fuller*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

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. Ser780 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 Asp780 >Ser780 >Ala780. Affinity-purified antibody against a peptide containing phospho-Ser780 recognizes wild-type Kex2p but not S780A Kex2p, showing that Ser780 is phosphorylated in vivo,phosphorylation of Ser780 is up-regulated by cell wall-damaging drugs. Finally, mutation of Ser780 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.

Original languageEnglish (US)
Pages (from-to)495-509
Number of pages15
JournalMolecular biology of the cell
Volume24
Issue number4
DOIs
StatePublished - Feb 15 2013

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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