Immunofluorescent localization of 100K coated vesicle proteins.
A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.
Robinson
M S
MS
Pearse
B M
BM
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D009940:Organoids / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis
D002966:Clathrin; D010446:Peptide Fragments