Brain Clathrin: Studies of Its Ultrastructural Assemblies

Abstract

Clathrin p u r i h d to a high dzgrec of hoinogzncity showed thz presencz of accompanying polypzptidcs of lowsr molccular wcights and asscmblitd into baskcls or c a g a when thc pH of its solution was adjusted from pH 7.5 to 6.5. Brief chyinotrypsin treatment of this clathrin preparation at pH 6.5 cleaved clathrin-associated proteins rendzring clathrin unablc to rcform baskets. Hydrolysis of clathrin-associated proteins did not afYect clathrin's capacity to polymerize into open latticzs or to bind actin and a-actinin from solution. W1ii.n open latticcs formzd, th; turbidity of the solution rose to lzvels that wcr2 higha than t1ios;'produced when cages or baskets wirc: form2d by native clathrin. In both native or enzyme-treated clathrin, turbidity increase was inhibited by salts. The addition of ccrtain cytoskzlctal-disrupting agents, such a s chlorpromazinit and imipraminz, incrzascd clathrin's turbidit y but did not r.:sult in formation of baskets. Colchicinz and cytochalasin B did not affect turbidity or t h s asszmbly o f cagzs. Thz addition of incrcasing ainounls of vinblastin,: rcsulted in the prxipitation of larger amounts of clatlirin which, after solubilization, r=taincd its ability to polymierize into baskets. I t sJems thal clathrin (exists as a complex with other protein moleculcs, clatlirin-associatzd protdins, that inodulatz tht: cxtznt of polyincrization and asscmbly of clathrin into charactcristic structurzs.

Clathrin heavy chain, light chain interactions.

“…A characteristic of native clathrin is its ability to assemble into polygonal cage structures morphologically identical to those observed on coated vesicles. In all previous studies with clathrin heavy chains, the light chains were selectively removed by mild proteolysis using elastase (Kirchhausen and Harrison, 1981;Schmid et al, 1982) or chymotrypsin (Lisanti et al, 1981(Lisanti et al, , 1982a. Such heavy chains predominantly formed large irregular aggregates under standard reassembly conditions, suggesting that the light chains were required for the formation of proper cages.…”

Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.

“…The demonstration of CAP2 phosphorylation offers exciting possibilities for the regulation of clathrin assembly/disassembly and coated vesicle-organellar interactions, since the CAPs are known to protrude from the surface of the coated vesicle (26) and may play a role in triskelion assembly/disassembly (27,28). The requirement for a nonvesicle-associated effector suggests that cytosolic components may be found that can produce the same effect as polylysine or histones and regulate phosphorylation of CAP2.…”

Phosphorylation Characteristics of Brain Clathrin‐Coated Vesicle Endogenous Proteins

“…One of the substrates, pp50, is a component of the assembly polypeptide complex (Zaremba and Keen, 1985;Robinson and Pearse, 1986), which has a role in the formation of clathrin lattices. CAP2 is involved in the maintenance of the clathrin structure and in the interaction of clathrin with other proteins (Lisanti et al, 1982;Schmid and Rothman, 1985). The discovery of conditions resulting in phosphate incorporation into CAP2 prompted us to investigate the properties of the kinase(s) responsible for phosphorylating both pp50 and CAP2 and to define the enzyme(s) by their specific activation or inhibitors.…”