Identification and characterization of a testis-specific isoform of a chaperonin in a moth, Heliothis virescens

Abstract

Two relatively abundant proteins having subunit molecular weights of 60, 000 and 63, 000 (p60 and p63, respectively) have been purified as a 16 to 18 S complex from sperm mitochondria of a moth, Heliothis virescens. Although the function of these proteins had heretofore not been established, interest in the p63 polypeptide stemmed from its sperm-specific expression and its striking occurrence as a net charge variant among several insect species surveyed, using two-dimensional gel electrophoresis. Genomic and cDNA clones corresponding to the p63 protein have now been isolated and their sequencing has revealed extensive amino acid sequence identity with both the Escherichia coli GroEL protein and its eukaryotic homologues, the chaperoning. Immunoblot studies with a Tetrahymena chaperonin antiserum demonstrated that the p60 protein, which is expressed in all cell types, is structurally related to p63 and is itself a chaperonin subunit. While the chaperonin complex from Heliothis sperm shares certain properties with GroEL, including the ability to hydrolyze ATP and organization of its subunits into a seven-member ring, electron microscopic analysis revealed that its higher-order structure differed from GroEL (and other lower eukaryotic chaperonins) in that the native particle comprises one such ring rather than a doublet. It is not yet known whether the two chaperonin isoforms coexpressed in moth sperm assemble separately or give rise to hybrid particles. In either case, the existence of multiple chaperonin subunits in sperm leaves open the possibility that some aspect of mitochondrial biogenesis that is dependent upon the activity of these proteins is qualitatively or quantitatively different in this cell type.