Structural characterization of labeled clathrin and coated vesicles.
Clathrin (8 S) and coated vesicles have been covalently labeled by using the sulfhydryl-labeling fluorescent probe N-(1-anilinonaphthalene)maleimide. A large increase in energy transfer from Trp to anilinonaphthalene (AN) residues was observed in clathrin in the pH range approximately 6.5-6.0, where the rate of clathrin self-association increased rapidly. The change in energy transfer was indicative of a conformational rearrangement, which could be responsible for the initiation of the clathrin self-association reaction to form coat structure. The AN label was found in both the coat and membrane proteins after dissociation of coated vesicles at pH 8.5. The labeled coat and membrane proteins readily recombined to form coated vesicles after reducing the pH to 6.5, indicating that the labeling did not interfere with the ability of clathrin to self-associate and interact with uncoated vesicles to form coat structure. A comparison of the AN fluorescence with the Coomassie blue pattern after electrophoresis in sodium dodecyl sulfate-gels revealed that a 180,000-Da protein (clathrin) was mainly labeled in coated vesicles, while a 110,000-Da protein was also strongly labeled in uncoated vesicles. AN-labeled baskets and coated vesicles have been prepared. Trypsin digestion reduced the sedimentation rate of baskets from 150 S to 120 S and of coated vesicles from 200 S to 150 S. Gel electrophoresis of baskets and coated vesicles showed extensive conversion of clathrin (Mr 180,000) to a product of Mr approximately equal to 110,000, suggesting equivalent structural organization of the coat in coated vesicles as in baskets. In both cases, the peptide(s) released from the vesicles by digestion were essentially free of fluorescent label. In the case of the uncoated vesicles, tryptic digestion released most of the proteins remaining after coat removal.
D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002966:Clathrin; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D006863:Hydrogen-Ion Concentration; D008301:Maleimides; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin
D002966:Clathrin; D008301:Maleimides; D013438:Sulfhydryl Compounds; C010854:N-(1-anilinonaphthalyl)-4-maleimide; D014357:Trypsin