The Truncated Form of the Bacterial Heat Shock Protein ClpB/HSP100 Contributes to Development of Thermotolerance in the Cyanobacterium Synechococcus sp. Strain PCC 7942

Abstract

ClpB is a highly conserved heat shock protein that is essential for thermotolerance in bacteria and eukaryotes. One distinctive feature of all bacterial clpB genes is the dual translation of a truncated 79-kDa form (ClpB-79) in addition to the full-length 93-kDa protein (ClpB-93). To investigate the currently unknown function of ClpB-79, we have examined the ability of the two different-sized ClpB homologues from the cyanobacterium Synechococcus sp. strain PCC 7942 to confer thermotolerance. We show that the ClpB-79 form has the same capacity as ClpB-93 to confer thermotolerance and that the ClpB-79 protein contributes ca. one-third of the total thermotolerance developed in wild-type Synechococcus, the first in vivo demonstration of a functional role for ClpB-79 in bacteria.Changing environmental conditions elicit the synthesis of new types of cellular proteins in all organisms. This conserved molecular response is best exemplified by high-temperature stress, during which specific groups of polypeptides known as heat shock proteins (HSPs) are rapidly induced. Many of these HSPs are now known to function as molecular chaperones and are part of larger protein families that include constitutive members. Protein denaturation and aggregation are the major types of cellular damage that result from high temperatures, and HSP chaperones respond by preventing aggregation, assisting refolding, and targeting misfolded protein for degradation (13). The activity of such chaperones is essential for cell survival during heat shock and for subsequent recovery.ClpB is a heat shock-inducible representative of the HSP100/Clp family of oligomeric ATPases. The family can be divided into several types based on specific sequence signatures and other structural characteristics (17). Most member proteins are large (79 to 105 kDa) and contain two distinct nucleotide-binding domains separated by a spacer region of variable length (ClpA-E), whereas some are smaller (50 kDa) and have only one such domain (ClpX and ClpY). The various HSP100/Clp proteins are thought to function as molecular chaperones, with a common mechanism of dismantling multimeric protein complexes, as shown for ClpA and -X in Escherichia coli (7,22).ClpB is an HSP found in almost all organisms studied to date. Separate nuclear genes encode two different-sized ClpB proteins in yeast, a 78-kDa protein localized in mitochondria (6) and a 104-kDa protein localized primarily in the cytosol (16). Both ClpB proteins function as molecular chaperones. Along with DnaK (HSP70), mitochondrial ClpB helps prevent protein denaturation and aggregation at high temperatures (18, 19), whereas cytosolic ClpB (HSP104) disassembles large protein aggregates that accumulate at extreme high temperatures (14). Cytosolic ClpB also acts in concert with DnaK or DnaJ to promote the refolding of the once aggregated polypeptides (4). Such functions are thought to underlie the necessity of cytosolic ClpB for the acquisition of thermotolerance in yeast (16), which is the tolerance developed to a normally...

Genetic Analysis Reveals Domain Interactions of Arabidopsis Hsp100/ClpB and Cooperation with the Small Heat Shock Protein Chaperone System

“…Proteolytic removal of the C terminus in V813M supports the requirement of this domain for function, which as stated before, may involve not only hexamerization, but also effector binding (Smith et al, 1999;Strub et al, 2003). Although our screen was not performed to saturation, it is notable that no loss-of-function suppressors were obtained in the N-terminal domain, which has also not been associated with an essential function in other organisms (Clarke and Eriksson, 2000;Beinker et al, 2002;Mogk et al, 2003b).…”

“…Biochemical studies using both in vitro assays for ATP hydrolysis and protein disaggregation (chaperone activity), along with in vivo assays for thermotolerance, have also begun to dissect functional domains of Hsp100/ClpB (Smith et al, 1999;Barnett et al, 2000;Clarke and Eriksson, 2000;Schirmer et al, 2001;Beinker et al, 2002;Cashikar et al, 2002;Mogk et al, 2003b;Strub et al, 2003). However, details of the Hsp100/ClpB reaction cycle are unknown.…”

“…The ATPase activity of both NBDs, as well as an intact coiled-coil domain, are essential for chaperone activity and in vivo thermotolerance (Kim et al, 2000a;Schirmer et al, 2001;Hattendorf and Lindquist, 2002b;Mogk et al, 2003b). A majority of data indicate that deletion of the N-terminal domain does not impair measured activities, and its function remains unknown (Clarke and Eriksson, 2000;Beinker et al, 2002). The C-terminal domain is required for activity, but because deletion of this domain prevents hexamerization, which is required for ATPase activity, its direct role is not revealed by such experiments.…”

Interplay between Heat Shock Proteins HSP101 and HSA32 Prolongs Heat Acclimation Memory Posttranscriptionally in Arabidopsis

“…S1). The NTD was shown to be dispensable for chaperone activity in Escherichia coli and Thermus thermophilus (Beinker et al, 2002;Mogk et al, 2003) and for thermotolerance in cyanobacteria and yeast (Clarke and Eriksson, 2000;Hung and Masison, 2006). However, work on E. coli ClpB showed that truncation of the NTD causes severe defects in molecular chaperone activity in vitro (Barnett et al, 2000;Li and Sha, 2003).…”

N-Terminal Domain of Yeast Hsp104 Chaperone Is Dispensable for Thermotolerance and Prion Propagation but Necessary for Curing Prions by Hsp104 Overexpression

“…These truncated proteins function well in protein disaggregation and the role of the NTD in ClpB function is still speculative (Clarke and Eriksson 2000;Beinker et al 2002;Mogk et al 2003;Chow et al 2005). The NTD of ClpB can affect interactions with aggregated model substrates (Beinker et al 2002;Tek and Zolkiewski 2002;Mogk et al 2003;Barnett et al 2005), but this activity clearly is not essential for ClpB to recognize and act on protein aggregates.…”