Purification and characterization of a novel 13 S hetero-oligomeric protein complex that stimulates in vitro Golgi transport.
Intracellular protein traffic involves a tightly regulated series of events in which a membrane-bounded vesicles bud from one compartment and are specifically targeted to the next compartment, where they dock and fuse. A cell-free system that reconstitutes vesicle trafficking between the cis and medial Golgi cisternae has been used previously to identify several proteins involved in vesicular transport (N-ethylmaleimide-sensitive fusion protein, soluble N-ethylmaleimide-sensitive fusion protein attachment proteins, p115, and p16); however, these factors are insufficient to drive the transport reaction. We have used a modified version of this in vitro intra-Golgi transport assay to guide purification of a new transport-stimulating activity. The active component is a 13 S hetero-oligomeric complex consisting of at least five polypeptides (approximately 110, 109, 90, 82, and 71 kDa), which we term Golgi transport complex (GTC). Hydrodynamic properties suggest that GTC is approximately 800 kDa and nonglobular. We obtained peptide sequence information from the 90-kDa subunit (GTC-90) that allowed us to identify a number of GTC-90 cDNAs. Comparison of these cDNAs with one another and with the genomic sequence suggests that the GTC-90 mRNA is alternatively spliced. Anti-GTC-90 antibodies inhibit the in vitro Golgi transport assay, confirming the functionality of the purified complex. Subcellular fractionation indicates that GTC-90 exists in both membrane and cytosolic pools, with the cytosolic pool associated exclusively with the GTC complex. The membrane-associated pool of GTC-90 is localized to the Golgi apparatus.
Walter
D M
DM
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Paul
K S
KS
Waters
M G
MG
D033942:Adaptor Proteins, Vesicular Transport; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012333:RNA, Messenger / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; C115632:COG5 protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger