Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF058718
Several studies have shown that the assay signal is not affected by removal of ADP-ribosylation factor [13, 37, 38], which is required for vesicle formation [9], suggesting that the transfer of VSV-G protein from donor to acceptor Golgi occurs via a partial transport reaction encompassing only the membrane docking and fusion steps
We found that assays performed in the presence of functionally saturating amounts of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and p115 still required the addition of cytosol for optimal activity, suggesting that at least one other factor was required, as had been previously suggested [12]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF058718. Several studies have shown that the assay signal is not affected by removal of ADP-ribosylation factor [13, 37, 38], which is required for vesicle formation [9], suggesting that the transfer of VSV-G protein from donor to acceptor Golgi occurs via a partial transport reaction encompassing only the membrane docking and fusion steps In this regard, it is noteworthy that the four proteins purified based on their activity in this in vitro system (NSF, SNAPs, p115, and p16) impact on the docking or fusion phases of transport, as shown by both biochemical studies [10, 11, 13, 14, 22] and genetic analyses of their homologs in yeast [15, 39]. Removal of Rab proteins, which are generally thought to be involved in membrane docking [40], inhibits the in vitro assay [41]
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