The products of the groES and groEL genes of Escherichia coli, constituting the groE operon, are known to be required for growth at high temperature (42°C) and are members of the heat shock regulon. Using a genetic approach, we examined the requirement for these gene products for bacterial growth at low temperature (17 to 30°C). To do this, we constructed various groES groEL heterodiploid derivative strains. By inactivating one of the groE operons by a polar insertion, it was shown by bacteriophage P1 transduction that at least one of the groE genes was essential for growth at low temperature. Further P1 transduction experiments with strains that were heterodiploid for only one of the groE genes demonstrated that both groE gene products were required for growth at low temperature, which suggested a fundamental role for the groE proteins in E. coli growth and physiology.The groES (mopA) and groEL (mopB) genes of Escherichia coli form an operon located at 94.2 min on the standard genetic map (2). They were first defined by mutations affecting the morphogenesis of several bacteriophages, including X, T4, and T5 (see reference 6 for a review). Both of the groE gene products have been shown to be essential for bacteriophage X head assembly (6)(7)(8)33) and for bacteriophage T5 tail assembly (8,40). In addition, the groEL gene product has been shown to be required for proper T4 head assembly (6,7,29,32). Some alleles of both genes were subsequently shown to also result in thermosensitive bacterial growth at 42°C, affecting both DNA and RNA synthesis (ts mutants; 36). Both the bacterial temperature-sensitive phenotype and inability to propagate bacteriophage A always contransduced, which demonstrated that the groE gene products are required for bacterial growth at least at high temperature (7). Although the exact role of these gene products in cell physiology remains to be determined, several of their properties are known at the physiological and molecular levels. The groES and groEL genes code for 10,368-and 57,259-Mr acidic polypeptides, respectively, found at high intracellular levels (about 2% of total cell proteins at 37°C; 12, 26, 33). Furthermore, as members of the heat shock regulon, the intracellular levels of their products increase with temperature through a positive transcriptional control exerted by the rpoH (U32) gene product (10).The products of some of the heat shock genes are either totally indispensable (e.g., rpoD, which codes for the Cr70 subunit of E. coli RNA polymerase; 26), dispensable (e.g., lon, which codes for an ATP-dependent protease [22,26]

Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s). The Saccharomyces cerevisiae HSP70 family is comprised of eight members. Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family. We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species. Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed. Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins. Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts. HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles. The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago. In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S. cerevisiae, more recent duplication events have given rise to subfamilies within the major groups. The S. cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date. This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups.

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The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb-ribosome interaction was characterized. Incorporation of the aminoacyl-tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross-linked to nascent chains containing a modified lysine residue with a photoactivatable cross-linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome-nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins.

Diverse studies of three cytoplasmic proteins of Escherichia coli‐‐SecB, trigger factor and GroEL‐‐have suggested that they can maintain precursor proteins in a conformation which is competent for membrane translocation. These proteins have been termed ‘chaperones’. Using purified chaperone proteins and precursor protein substrates, we find that each of these chaperones can stabilize proOmpA for translocation and for the translocation‐ATPase. These chaperones bind to proOmpA to form isolable complexes. SecB and GroEL will also form complexes with another exported protein, prePhoE. In contrast, these chaperones do not form stable complexes with a variety of soluble proteins such as SecA protein, bovine serum albumin, ovalbumin or ribonuclease A. While chaperones may transiently interact with soluble proteins to catalyze their folding, the stable interaction between chaperones and presecretory proteins, maintaining an open conformation which is essential for translocation, may commit these proteins to the secretion pathway.