CHAPERONIN FILAMENTS - THEIR FORMATION AND AN EVALUATION OF METHODS FOR STUDYING THEM

Chaperonins are multisubunit protein complexes that can be isolated fr om cells as high-molecular-weight structures that appear as double rin gs in the electron microscope. We recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon Sulfolobus s hibatae, when incubated at physiological temperatures in the presence of ATP and Mg2+, stacked into filaments; we hypothesized that these fi laments are related to filaments seen inside S. shibatae cells and tha t chaperonins exist as filaments in vivo (J. D. Trent ct al, 1997, Pro c. Natl. Acad. Sci. USA 94, 5383-5388). This paper elucidates the cond itions under which we have observed S. shibatae chaperonins to form fi laments and evaluates native polyacrylamide gel electrophoresis (PAGE) , TEM, spectrophotometry, and centrifugation as methods for studying t hese filaments. We observed that in the presence of Mg2+ combined with ATP, ADP, ATP gamma S, or GTP, native PAGE indicated that chaperonin subunits assembled into double rings and that the conformation of thes e double rings wars effected by nucleotide binding, but we saw no indi cation of chaperonin filament formation. Under these same conditions, however, TEM, spectroscopy, and centrifugation methods indicated that chaperonin subunits and double rings had assembled into filaments. We determined that this discrepancy in the representation of the chaperon in structure was due to the native PAGE method itself. When we exposed chaperonin filaments to the electrophoretic held used in native PAGE, the filaments dissociated into double rings. This suggests that TEM, spectrophotometry, and centrifugation are the preferred methods for st udying the higher-order structures of chaperonins, which are likely to be of biological significance. (C) 1998 Academic Press.