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.

A yeast two-hybrid screen reveals a strong interaction between the Legionella chaperonin Hsp60 and the host cell small heat shock protein Hsp10

“…For yeast samples, 10 8 pelleted cells (800 × g for 5 min) were resuspended in 200 μL of sample buffer containing the α-yeast protease inhibitor cocktail (Sigma), and mechanically broken by adding ~100 μL of acid-washed and baked glass beads (BT-5 high impact beads, 40-50 μm diameter, Supply America Company Inc., Norfolk, Virginia) and vortex mixing at 4°C for 15 min. Samples were then boiled for 5 min, unbroken cells and cell wall debris pelleted at 15,000 × g, and 10 μL of the supernatant per lane subjected to SDS-PAGE.…”

“…For example, Group I chaperonins are proteins found in bacteria (known as Cpn60, GroEL, Hsp60, or HtpB), and in eukaryotic organelles such as chloroplasts and mitochondria (known as CCT, TRiC or c-cpn) [4]. A comparison of the aligned amino acid sequences of the eukaryotic chaperonins and bacterial chaperonins (including HtpB) revealed that these proteins have high degrees of amino acid homology that sometimes exceed 70% [4][5][6][7][8]. To function properly in protein folding, bacterial or eukaryotic chaperonins must work in conjunction with a small heat shock protein (~10 kDa) known as Hsp10 or Cpn10 (also known as GroES in bacteria and HtpA in L. pneumophila) [4,9,10].…”

“…To function properly in protein folding, bacterial or eukaryotic chaperonins must work in conjunction with a small heat shock protein (~10 kDa) known as Hsp10 or Cpn10 (also known as GroES in bacteria and HtpA in L. pneumophila) [4,9,10]. The amino acid sequences homology of the eukaryotic and bacterial co-chaperonins is also highly conserved among Group I chaperonins family [8,11,12]. Surprisingly, in vitro protein binding assays shows that eukaryotic chaperonins Cpn60 bind to bacterial, mitochondrial, and chloroplast Cpn10 homologs with comparable affi nities, suggesting that chaperonins from bacteria can interact with co-chaperonin from eukaryotic cells and vice versa [8].…”

“…The amino acid sequences homology of the eukaryotic and bacterial co-chaperonins is also highly conserved among Group I chaperonins family [8,11,12]. Surprisingly, in vitro protein binding assays shows that eukaryotic chaperonins Cpn60 bind to bacterial, mitochondrial, and chloroplast Cpn10 homologs with comparable affi nities, suggesting that chaperonins from bacteria can interact with co-chaperonin from eukaryotic cells and vice versa [8].…”

“…In this study, based on the facts that (i) there is a high degree of homology between eukaryotic and bacterial co-chaperonins, (ii) bacterial chaperonin can interact with eukaryotic co-chaperonin [8], we hypothesized that HtpB could recruit the host cell Hsp10 as co-chaperonin in order to (i) appropriately interacts with SAMDC and to (ii) mediate mitochondrial attraction and actin microfi laments reorganization in host cells [3,18]. Indeed, in this study, using yeast two-hybrid (Y2H) screen of HeLa cell cDNA library, we showed that HtpB strongly interacts with the small heat shock protein Hsp10 of HeLa.…”