p230 is associated with vesicles budding from the trans-Golgi network.
Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using alpha 2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTP gamma S or AlF4- resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4-, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (approximately 10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.
Gleeson
P A
PA
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Victoria, Australia. pag@cobra.path.monash.edu.au
Anderson
T J
TJ
Stow
J L
JL
Griffiths
G
G
Toh
B H
BH
Matheson
F
F
D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D001323:Autoantibodies / Q000378:metabolism*; D001324:Autoantigens; D020126:Brefeldin A; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology; D004285:Dogs; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000648:ultrastructure*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron
D017607:Aluminum Compounds; D001323:Autoantibodies; D001324:Autoantigens; D003517:Cyclopentanes; C098772:GOLGA4 protein, human; D008565:Membrane Proteins; D020126:Brefeldin A; C050992:tetrafluoroaluminate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D005459:Fluorides