Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly.
Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg2+, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES--1 m M EGTA--1 mM MgCl2--0.02% NaN3, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg2+ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl2 prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl2 is raised to 10 mM, reassembly occurs. The results suggest that Mg2+ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg2+ (10 microM--10 mM) increases reassembly whereas chelation of Mg2+ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl2 do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.
D001692:Biological Transport; D002021:Buffers; D004533:Egtazic Acid / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D009940:Organoids / Q000032:analysis; D011487:Protein Conformation / Q000187:drug effects; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D014325:Tromethamine / Q000494:pharmacology
D002021:Buffers; D011506:Proteins; D014325:Tromethamine; D004533:Egtazic Acid; D008274:Magnesium