An acidic sequence of a putative yeast Golgi membrane protein binds COPII and facilitates ER export

EMBO J. 2001 Dec 3;20(23):6742-50. doi: 10.1093/emboj/20.23.6742.

Abstract

We previously identified Sys1p as a high copy number suppressor of Ypt6 GTPase-deficient yeast mutants that are defective in endosome-to-Golgi transport. Here, we show that Sys1p is an integral membrane protein that resides on a post-endoplasmic reticulum (ER) organelle(s). Affinity studies with detergent- solubilized yeast proteins showed that the C-terminal 53 amino acid tail of Sys1p binds effectively to the cytoplasmic Sec23p-Sec24p COPII subcomplex. This binding required a di-acidic Asp-Leu-Glu (DXE) motif, previously shown to mediate efficient ER export of the vesicular stomatitis virus glycoprotein in mammalian cells. In Sys1p, a Glu-Leu-Glu (EXE) sequence could not substitute for the (DXE) motif. Mutations of the (DXE) sequence resulted in ER retention of approximately 30% of the protein at steady state, whereas addition of the Sys1p tail to an ER-resident membrane protein led to an intracellular redistribution of the chimeric protein. Our study demonstrates for the first time that, in yeast, a di-acidic sequence motif can act as a sorting signal for cargo selection during the formation of transport vesicles at the ER by direct binding to COPII component(s).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • COP-Coated Vesicles / chemistry
  • COP-Coated Vesicles / metabolism*
  • Cell Membrane / metabolism
  • Cloning, Molecular
  • Cytoplasm / metabolism
  • Dimerization
  • Endopeptidases / metabolism
  • Endoplasmic Reticulum / metabolism*
  • Epitopes
  • Escherichia coli / metabolism
  • GTP Phosphohydrolases / metabolism
  • Glutathione Transferase / metabolism
  • Golgi Apparatus / metabolism*
  • Intracellular Membranes / metabolism*
  • Membrane Glycoproteins*
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Monomeric GTP-Binding Proteins / metabolism
  • Mutation
  • Plasmids / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Transport
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Subcellular Fractions
  • Temperature
  • Time Factors
  • Vesicular Transport Proteins
  • Viral Envelope Proteins / genetics

Substances

  • Epitopes
  • G protein, vesicular stomatitis virus
  • Membrane Glycoproteins
  • Membrane Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Sys1 protein, S cerevisiae
  • Vesicular Transport Proteins
  • Viral Envelope Proteins
  • Glutathione Transferase
  • Endopeptidases
  • GTP Phosphohydrolases
  • Monomeric GTP-Binding Proteins
  • YPT6 protein, S cerevisiae

Associated data

  • GENBANK/AJ132966
  • PDB/12CA5
  • PDB/9E10