Abstract
The eukaryotic, cytoplasmic chaperonin, CCT, is essential for the biogenesis of actin- and tubulin-based cytoskeletal structures. CCT purifies as a doubly toroidal particle containing two eight-membered rings of approximately 60-kDa ATPase subunits, each encoded by an essential and highly conserved gene. However, immunofluorescence detection with subunit-specific antibodies has indicated that in cells CCT subunits do not always co-localize. We report here that CCT ATPase activity is highly dependent on K+ ion concentration and that in cell extracts, at physiological levels of K+ and ATP, there is considerable dissociation of CCT to a smaller oligomeric structure and free subunits. This dissociation is consequent to ATP hydrolysis and is readily reversed on removal of ATP. The ranking order for ease with which subunits can exit the chaperonin particle correlates well with the length of a loop structure, identified by homology modeling, in the intermediate domain of CCT subunits. K+-ATP-induced disassembly is not an intrinsic property of purified CCT over a 40-fold concentration range and requires the presence of additional factor(s) present in cell extracts.
Highlights
Domain, which contains binding sites for unfolded proteins and GroES [7]
Immunoprecipitations targeted at the majority of CCT subunits from cell extracts yielded the “core” hetero-oligomeric particle, immunoprecipitation in the presence of intracellular levels of ATP tended to yield only the subunit targeted for precipitation
The data presented below were obtained from cell extracts and CCT prepared from the ND7/23 cell line unless otherwise stated
Summary
Domain, which contains binding sites for unfolded proteins and GroES [7]. Folding substrate binding, ATP binding, GroES binding, folding substrate release, and ATP hydrolysis by GroEL subunits is a highly co-ordinated sequence of events [8], and this, together with their forming the enclosed central folding cavity, is likely to make the double-toroidal structure formed by the GroEL subunits essential to most of the protein folding activities of this chaperonin in vivo [9]. Our own data, based on immunofluorescence microscopy and various biochemical analyses, utilizing antibodies specific to each of the eight CCT subunits, have consistently indicated that substantial amounts of CCT subunits are not always components of the hetero-oligomeric chaperonin particle in the environment of the cell [28, 32, 33]. Immunoprecipitations targeted at the majority of CCT subunits from cell extracts yielded the “core” hetero-oligomeric particle, immunoprecipitation in the presence of intracellular levels of ATP tended to yield only the subunit targeted for precipitation (see Fig. 8 in Ref. 28) These observations were made in cell extracts prepared in a buffer containing 90 mM KCl. We report here that at intracellular levels of Kϩ ions and ATP, there is substantial disassembly of the CCT chaperonin to free subunits contingent upon the Kϩ-dependent hydrolysis of ATP. This disassembly is readily reversed upon removal of ATP and is dependent on (an) additional factor(s) present in cell extracts but removed during purification of the CCT particle
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