Purification and characterization of clathrin-coated vesicles from Chlamydomonas.
Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas reinhardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter +/- SD of 95 +/- 17 nm, n = 300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.
Denning
G M
GM
Department of Biochemistry, University of Iowa, Iowa City 52242.
Fulton
A B
AB
D015153:Blotting, Western; D002696:Chlamydomonas / Q000032:analysis / Q000648:ultrastructure*; D002848:Chromatography, DEAE-Cellulose; D002966:Clathrin / Q000032:analysis*; D003034:Coated Pits, Cell-Membrane / Q000032:analysis / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000648:ultrastructure*; D010455:Peptides / Q000302:isolation & purification; D013194:Staining and Labeling
D002966:Clathrin; D010455:Peptides