Sec34 is implicated in traffic from the endoplasmic reticulum to the Golgi and exists in a complex with GTC-90 and ldlBp.
Sec34p/Grd20p has been implicated in endoplasmic reticulum (ER)-to-Golgi transport and/or post-Golgi trafficking events and exists in a protein complex consisting of at least eight subunits in yeast. Although the mammalian counterpart (Sec34) of Sec34p has been molecularly identified, its role and interacting partners remain undefined. In this study, we have prepared antibodies specifically against the recombinant N-terminal fragment of Sec34 that recognize a polypeptide of about 93 kDa and label the Golgi apparatus. In a well-characterized semi-intact cell assay that reconstitutes transport of the envelope glycoprotein (VSVG) of vesicular stomatitis virus from the ER to the Golgi apparatus, anti-Sec34 antibodies inhibited the transport in a dose-dependent manner. The inhibition by anti-Sec34 antibodies could be neutralized by a noninhibitory amount of the antigen. Large-scale immunoprecipitation of rat liver cytosol with immobilized anti-Sec34 antibodies has co-immunoprecipitated GTC-90 and ldlBp, two peripheral Golgi proteins previously shown to exist in separate protein complexes. Two mammalian homologues (Dor1 and Cod1) of the yeast Sec34 complex were similarly recovered in the Sec34 immunoprecipitates. When expressed in transfected cells, epitope-tagged ldlCp and Cod2 were co-immunoprecipitated with anti-Sec34 antibodies with efficiencies comparable to that observed for tagged ldlBp, Dor1, and Cod1. Direct interactions of Sec34 with ldlBp and ldlCp were further demonstrated in vitro. These results suggest that Sec34, GTC-90, and ldlBp/ldlCp are part of the same protein complex(es) that regulates diverse aspects of Golgi function, including transport from the ER to the Golgi apparatus.
Loh
Eva
E
Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore.
Hong
Wanjin
W
D018528:ATP-Binding Cassette Transporters; D033942:Adaptor Proteins, Vesicular Transport; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D000939:Epitopes; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D033921:Vesicular Transport Proteins
D018528:ATP-Binding Cassette Transporters; D033942:Adaptor Proteins, Vesicular Transport; C486489:COG1 protein, human; C401396:COG3 protein, S cerevisiae; C498528:COG3 protein, human; C115632:COG5 protein, human; C460371:COG6 protein, S cerevisiae; D002352:Carrier Proteins; D018076:DNA, Complementary; D000939:Epitopes; D008565:Membrane Proteins; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C406793:SPF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases