Purification and characterization of a late Golgi compartment from Saccharomyces cerevisiae.
We have devised a method for obtaining highly enriched membranes of a late yeast Golgi compartment, operationally defined by their containing the Kex2p protease, and generated four hybridoma cell lines that produced monoclonal antibodies directed against distinct Golgi membrane proteins (GMPs) (GMP36, GMP51, GMP77, and GMP95). Immunofluorescence and subcellular fractionation data indicated that, of the four GMPs analyzed, only GMP51 exhibited essentially an absolute colocalization with Kex2p. Also, as in the case of Kex2p, retention of GMP51 in yeast Golgi membranes was dependent on clathrin function. In contrast, the remaining three GMPs exhibited substantial, but not absolute, colocalization with Kex2p. The collective data are most consistent with a model where GMP36, GMP77, and GMP95 are present in all Kex2p-containing membranes, but Kex2p is present in only a subpopulation of membranes that contain these GMPs, thereby suggesting that either these particular GMPs exhibit overlapping distributions in compartments of the yeast Golgi complex or are also present in non-Golgi compartments. These findings are not consistent with the view that resident yeast Golgi proteins are generally restricted to a specific Golgi subcompartment, but they are consistent with the view that Golgi compartmental identity is determined by the relative mixtures of Golgi proteins that reside within individual cisternae.
Department of Cell Biology, University of Alabama at Birmingham 35294-0005.
E A
EA
Whitters
T P
TP
McGee
V A
VA
Bankaitis
D000818:Animals; D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000032:analysis; D002458:Cell Fractionation; D002501:Centrifugation, Zonal; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000032:analysis*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure*; D006825:Hybridomas; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice / Q000276:immunology; D043484:Proprotein Convertases; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000032:analysis*
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae