title: Differential effects of co-chaperonin homologs on cpn60 oligomers.
abstract: In this study, we have investigated the relationship between chaperonin/co-chaperonin binding, ATP hydrolysis, and protein refolding in heterologous chaperonin systems from bacteria, chloroplast, and mitochondria. We characterized two types of chloroplast cpn60 oligomers, ch-cpn60 composed of alpha and beta subunits (alpha(7)beta(7) ch-cpn60) and one composed of all beta subunits (beta(14) ch-cpn60). In terms of ATPase activity, the rate of ATP hydrolysis increased with protein concentration up to 60 microM, reflecting a concentration at which the oligomers are stable. At high concentrations of cpn60, all cpn10 homologs inhibited ATPase activity of alpha(7)beta(7) ch-cpn60. In contrast, ATPase of beta(14) ch-cpn60 was inhibited only by mitochondrial cpn10, supporting previous reports showing that beta(14) is functional only with mitochondrial cpn10 and not with other cpn10 homologs. Surprisingly, direct binding assays showed that both ch-cpn60 oligomer types bind to bacterial, mitochondrial, and chloroplast cpn10 homologs with an equal apparent affinity. Moreover, mitochondrial cpn60 binds chloroplast cpn20 with which it is not able to refold denatured proteins. Protein refolding experiments showed that in such instances, the bound protein is released in a conformation that is not able to refold. The presence of glycerol, or subsequent addition of mitochondrial cpn10, allows us to recover enzymatic activity of the substrate protein. Thus, in our systems, the formation of co-chaperonin/chaperonin complexes does not necessarily lead to protein folding. By using heterologous oligomer systems, we are able to separate the functions of binding and refolding in order to better understand the chaperonin mechanism.
authors.affiliation: Department of Biochemistry, The George Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69778, Israel.
authors.forename: Anat L
authors.initials: AL
authors.lastname: Bonshtien
authors.forename: Avital
authors.initials: A
authors.lastname: Parnas
authors.forename: Rajach
authors.initials: R
authors.lastname: Sharkia
authors.forename: Adina
authors.initials: A
authors.lastname: Niv
authors.forename: Itzhak
authors.initials: I
authors.lastname: Mizrahi
authors.forename: Abdussalam
authors.initials: A
authors.lastname: Azem
authors.forename: Celeste
authors.initials: C
authors.lastname: Weiss
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D018834:Chaperonin 60 / Q000378:metabolism*; D018833:Chaperonins / Q000737:chemistry / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D018834:Chaperonin 60; D000255:Adenosine Triphosphate; D018833:Chaperonins