The Mammalian Homolog of Yeast Sec13p Is Enriched in the Intermediate Compartment and Is Essential for Protein Transport from the Endoplasmic Reticulum to the Golgi Apparatus

Abstract

The role of COPII components in endoplasmic reticulum (ER)-Golgi transport, first identified in the yeastSaccharomyces cerevisiae, has yet to be fully characterized in higher eukaryotes. A human cDNA whose predicted amino acid sequence showed 70% similarity to the yeast Sec13p has previously been cloned. Antibodies raised against the human SEC13 protein (mSEC13) recognized a cellular protein of 35 kDa in both the soluble and membrane fractions. Like the yeast Sec13p, mSEC13 exist in the cytosol in both monomeric and highermolecular-weight forms. Immunofluorescence microscopy localized mSEC13 to the characteristic spotty ERGolgi intermediate compartment (ERGIC) in cells of all species examined, where it colocalized well with the KDEL receptor, an ERGIC marker, at 15؇C. Immunoelectron microscopy also localized mSEC13 to membrane structures close to the Golgi apparatus. mSEC13 is essential for ER-to-Golgi transport, since both the His 6 -tagged mSEC13 recombinant protein and the affinity-purified mSEC13 antibody inhibited the transport of restrictive temperature-arrested vesicular stomatitis virus G protein from the ER to the Golgi apparatus in a semi-intact cell assay. Moreover, cytosol immunodepleted of mSEC13 could no longer support ER-Golgi transport. Transport could be restored in a dose-dependent manner by a cytosol fraction enriched in the high-molecular-weight mSEC13 complex but not by a fraction enriched in either monomeric mSEC13 or recombinant mSEC13. As a putative component of the mammalian COPII complex, mSEC13 showed partially overlapping but mostly different properties in terms of localization, membrane recruitment, and dynamics compared to that of ␤-COP, a component of the COPI complex.

Two Mammalian Sec16 Homologues Have Nonredundant Functions in Endoplasmic Reticulum (ER) Export and Transitional ER Organization

“…At the stage of ER export, mammalian homologues have been described for Sec12, Sar1, Sec23, Sec24, Sec13, and Sec31 (Kuge et al, 1994;Paccaud et al, 1996;Tang et al, 1997Tang et al, , 1999Weissman et al, 2001;Stankewich et al, 2006), and all of these proteins seem to function similarly to their yeast counterparts. However, mammalian cells often have more isoforms of a given COPII component.…”

“…A typical cultured mammalian cell contains several hundred punctate tER sites, some of which are clustered in a juxtanuclear region near the Golgi, and others of which are distributed throughout the peripheral ER network (Bannykh and Balch, 1997;Tang et al, 1997;Hammond and Glick, 2000). These tER sites tend to be disrupted by conventional immunofluorescence procedures, but with an improved protocol they can be readily visualized using anti-COPII antibodies (Hammond and Glick, 2000).…”

An isoform of the Golgi t-SNARE, syntaxin 5, with an endoplasmic reticulum retrieval signal.

“…Most of the peripheral structures did not stain with either of these markers or any others that we have tried. These structures are reminiscent of ER exit sites, but none of the available antibodies (Tang et al, 1997) presently permit double labeling to confirm or deny this suggestion. We also do not yet know whether there is any of the short form of syntaxin 5 in these peripheral structures.…”

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

“…Serums were depleted of GST antibodies and affinity purified using GST-Nup85, GST-Nup37 or a mixture of GST-Nup160-N and GSTNup160-C immobilized on a NHS-activated columns. The others antibodies used were affinity-purified rabbit polyclonal antibodies directed against human Nup133 and Nup107 (Belgareh et al, 2001), Nup96/p87 serum affinitypurified by using recombinant Nup96/p87 (residues 1291-1482 of Nup196) immobilized on nitrocellulose (Fontoura et al, 1999), affinity-purified antiNup96 (directed against a synthetic peptide corresponding to aa 1743-1763 of Nup196) (Hase and Cordes, 2003), affinity-purified anti-Sec13 (Tang et al, 1997) and Sec31 (Tang et al, 2000), anti-Tpr (Kuznetsov et al, 2002), or anti-myc epitope (Euromedex, Souffelweyersheim, France); monoclonal antibodies anti-myc (clone 9E10; Sigma-Aldrich, St. Louis, MO), anti-GFP (clone 7.1 and 13.1; Roche Diagnostics, Indianapolis, IN), anti-SC-35 (Sigma-Aldrich), anti-cyclin B1 (Santa Cruz Biotechnology), anti-mitosin (BD Biosciences, San Jose, CA), and mAb414 (Babco, Richmond, CA); the autoimmune CREST serum was obtained from J.C. Courvalin (Institut J. Monod, Paris, France). To detect Giantin, the TA10 scFv (Nizak et al, 2003), obtained from F. Perez (Institut Curie, Paris, France), was used at 1/100 and mixed with a goat anti-myc antibody (Santa Cruz Biotechnology).…”