Hsp70s, ubiquitous molecular chaperones, function in a myriad of biological processes, modulating polypeptides' folding, degradation and translocation across membranes, as well as protein-protein interactions. This multitude of roles is not easily reconciled with the near conformity of biochemical activity of Hsp70s, an ATP-dependent client protein binding/release cycle. Much of the functional diversity of Hsp70s is driven by a diverse class of cofactors, Jproteins (also called Hsp40s). Often, multiple J-proteins function with a single Hsp70. Some target Hsp70 activity to clients at precise locations in cells; others bind client proteins directly, thereby delivering specific clients to Hsp70, directly determining their fate.In their native cellular environment, polypeptides are constantly at risk of attaining conformations that prevent them from functioning properly and/or cause them to aggregate into large, potentially cytotoxic complexes. Molecular chaperones guide the conformation of proteins throughout their lifetime, preventing their aggregation by protecting interactive surfaces against non-productive interactions. Through such interactions, they aid in the folding of nascent proteins as they are synthesized by ribosomes, drive protein transport across membranes, and modulate protein-protein interactions by controlling conformational changes1 , 2 . In addition to these roles under optimal conditions, stresses can exacerbate protein conformational problems (for example, heat shock causing protein unfolding; oxygen radicals causing oxidation and nitrosylation). Although in some cases chaperones can facilitate (re)folding, often such rejuvenation is not possible. In such cases, chaperones can facilitate degradation, either by simply preventing aggregation and thus keeping clients susceptible to proteolysis or by actively facilitating their transfer to proteolytic systems. These diverse functions of molecular chaperones typically involve iterative client binding and release cycles until the client has reached its final active conformation, or has entered the proteolytic system (Figure 1). Strikingly, Hsp70s, one of the most ubiquitous classes of chaperones, has been implicated in all of the biological processes mentioned above2 , 3 . This Review focuses on the means by which Hsp70 molecular chaperone machinery participates in such diverse cellular functions. Their functional diversity is remarkable considering that within and across species, Hsp70s have very high sequence identity. They share a single biochemical activity, an ATPdependent substrate binding and release cycle combined with client protein recognition, which is typically rather promiscuous. The answer to this apparent conundrum lies in the fact that Hsp70s do not work alone, but rather as "Hsp70 machines", collaborating with and h.h.kampinga@med.umcg.nl, ecraig@wisc.edu. regulated by a number of (co)chaperones and cofactors. Here, using examples from yeast and human, we discuss several such factors, particularly concentrating on how the ar...

Depletion of a subset of 70K stress proteins in yeast mutants shows that they are involved in the post-translational import of precursor polypeptides into both mitochondria and the lumen of the endoplasmic reticulum. The identification of such a basic function may explain the remarkable evolutionary conservation of the gene family encoding these proteins.

By analysis of a temperature-sensitive yeast mutant, a heat-shock protein in the matrix of mitochondria, mitochondrial hsp70 (Ssclp), is found to be involved both in translocation of nuclear-encoded precursor proteins across the mitochondrial membranes and in (re)folding of imported proteins in the matrix.

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.

No abstract

The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

No abstract

The primary base sequence ofthe protein coding regions of the four small heat shock genes of Drosophila nmlanogaster present at cytological locus 67B has been determined. A single open reading frame large enough to encode a small heat shock protein is found for each gene. The molecular weights of the predicted proteins are in good agreement with experimentally determined values obtained from gel electrophoresis. The predicted amino acid sequences of the four small heat shock genes show strildng homologies over =50% of their lengths. This region of extensive homology extends from about amino acid 85 to amino acid 195 out of a total of a2oo amino acids. Comparison of the predicted sequence with the known sequences of other proteins revealed a remarkable similarity between this region of homology and the corresponding region of mammalian a-crystaliin. The possible functional significance of this structural similarity is discussed.Heat shock and a variety of other stimuli result in a dramatic change in the pattern of gene expression in Drosophila melanogaster (for review, see ref.