title: Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum.
abstract: Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; bajajk@gene.com schekman@berkeley.edu.
authors.forename: Kanika
authors.initials: K
authors.lastname: Bajaj Pahuja
authors.affiliation: Division of Nephrology & Hypertension, Oregon Health and Science University, Portland, OR 97239;
authors.forename: Jinzhi
authors.initials: J
authors.lastname: Wang
authors.affiliation: Division of Nephrology & Hypertension, Oregon Health and Science University, Portland, OR 97239;
authors.forename: Anastasia
authors.initials: A
authors.lastname: Blagoveshchenskaya
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
authors.forename: Lillian
authors.initials: L
authors.lastname: Lim
authors.affiliation: Department of Biology, Indian Institute of Science Education and Research, Pune 411008, India; Bioinformatics Institute, Singapore 136781; Department of Biological Sciences, National University of Singapore, Singapore 117543.
authors.forename: M S
authors.initials: MS
authors.lastname: Madhusudhan
authors.affiliation: Division of Nephrology & Hypertension, Oregon Health and Science University, Portland, OR 97239;
authors.forename: Peter
authors.initials: P
authors.lastname: Mayinger
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; bajajk@gene.com schekman@berkeley.edu.
authors.forename: Randy
authors.initials: R
authors.lastname: Schekman
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D022181:COP-Coated Vesicles / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C416669:Sac1 protein, mammalian
keywords: 14-3-3; COPII vesicles; SAC1; adaptor protein; reconstitution