An acidic sequence of a putative yeast Golgi membrane protein binds COPII and facilitates ER export.
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).
Votsmeier
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Department of Molecular Genetics, Max Planck Institute for Biophysical Chemistry, D-37070 Göttingen, Germany.
Gallwitz
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D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D022181:COP-Coated Vesicles / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000939:Epitopes; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013696:Temperature; D013997:Time Factors; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins / Q000235:genetics
D000939:Epitopes; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C444081:Sys1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins; D005982:Glutathione Transferase; D010450:Endopeptidases; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins; C499307:YPT6 protein, S cerevisiae