The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage A DNA replication at all temperatures and for bacterial survival under certain conditions. DnaK, the bacterial heat shock protein hsp7O analogue and putative chaperonin, possesses a weak ATPase activity. Previous work has shown that ATP hydrolysis allows the release of various polypeptides complexed with DnaK. Here we demonstrate that the ATPase activity of DnaK can be greatly stimulated, up to 50-fold, in the simultaneous presence of the DnaJ and GrpE heat shock proteins. The presence of either DnaJ or GrpE alone results in a slight stimulation of the ATPase activity of DnaK. The action of the DnaJ and GrpE proteins may be sequential, since the presence of DnaJ alone leads to an acceleration in the rate of hydrolysis of the DnaK-bound ATP. The presence of GrpE alone increases the rate of release of bound ATP or ADP without affecting the rate of hydrolysis. The stimulation of the ATPase activity of DnaK may contribute to its more efficient recycling, and it helps explaln why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.The Escherichia coli dnaK gene product, the prokaryotic analogue of hsp70, the eukaryotic 70-kDa heat shock protein, participates in a variety of basic cellular functions: (i) survival of bacteria under different stress conditions, (ii) initiation of bacteriophage A and E. coli oriC-dependent DNA replication, (iii) regulation of cell division, (iv) modulation of proteolysis, (v) protein phosphorylation, and (vi) transport of proteins across membranes (reviewed in refs. 1-3). Such a broad spectrum of action suggests involvement of the DnaK protein in some general mechanisms crucial for the survival of the cell. Pelham (4) has suggested that the heat shock proteins belonging to the hsp70 family are involved in binding to the hydrophobic domains of other proteins, exposed either naturally or as a result of stressful conditions. Such binding and release, following ATP hydrolysis, may allow the disassembly of "dead-end" protein structures formed under stress conditions. In support of this hypothesis, we have recently shown that the DnaK protein protects E. coli RNA polymerase from heat inactivation by preventing its aggregation. In addition, in an ATP-dependent reaction, the DnaK protein can also dissolve the RNA polymerase aggregates formed at high temperature, leading to a complete restoration of RNA polymerase activity (5). Early evidence that ATP may be involved in hsp70 function was the observation that the E. coli DnaK protein has a weak ATPase activity (6). It was subsequently shown that members of the mammalian hsp70 family of proteins bind tightly to ATP cross-linked to an agarose matrix (7) and that ATP is required for release of hsp70 protein from nuclei (8). Similar results were obtained when ATP was added to complexes of hsc70 (a constitutive member of the hsp70 family) and p53 (an anti-oncogenic protein) (9),...

Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced cell death. Clues to its molecular mechanism of action may lay in the recently reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism. In acidic environments Hsp70 binds with high affinity and specificity to BMP, thereby facilitating the BMP binding and activity of acid sphingomyelinase (ASM). The inhibition of the Hsp70-BMP interaction by BMP antibodies or a point mutation in Hsp70 (Trp90Phe), as well as the pharmacological and genetic inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann-Pick disease (NPD) A and B-severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM-is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds that enter the lysosomal lumen by the endocytic delivery pathway.

Mammalian genes are highly heterogeneous with respect to their nucleotide composition, but the functional consequences of this heterogeneity are not clear. In the previous studies, weak positive or negative correlations have been found between the silent-site guanine and cytosine (GC) content and expression of mammalian genes. However, previous studies disregarded differences in the genomic context of genes, which could potentially obscure any correlation between GC content and expression. In the present work, we directly compared the expression of GC-rich and GC-poor genes placed in the context of identical promoters and UTR sequences. We performed transient and stable transfections of mammalian cells with GC-rich and GC-poor versions of Hsp70, green fluorescent protein, and IL2 genes. The GC-rich genes were expressed several-fold to over a 100-fold more efficiently than their GC-poor counterparts. This effect was not due to different translation rates of GC-rich and GC-poor mRNA. On the contrary, the efficient expression of GC-rich genes resulted from their increased steady-state mRNA levels. mRNA degradation rates were not correlated with GC content, suggesting that efficient transcription or mRNA processing is responsible for the high expression of GC-rich genes. We conclude that silent-site GC content correlates with gene expression efficiency in mammalian cells.

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.Both procaryotic and eucaryotic cells respond to a variety of stresses by accelerating the rate of synthesis of a group of proteins called heat shock proteins. This response in Escherichia coli is positively regulated by the product of the rpoH (htpR) gene, a32, which enables RNA polymerase to recognize promoters of heat shock genes (21). The heat shock genes all belong to the rpoH (htpR) regulon, are absolutely necessary for bacterial survival at high temperatures, and perform various, although not very well understood, functions in the cell. One of the processes in which the heat shock proteins are involved is cellular proteolysis. For example, the Lon protease belongs to the group of heat shock proteins (7,8). It is known that the accumulation of abnormal proteins leads to the induction of the rpoH (htpR)-dependent heat shock response (1, 12) and that aberrant proteins are frequently subject to proteolysis (13,14,28). It has been shown that, to a large extent, this proteolysis is rpoH (htpR) dependent; i.e., rpoH (htpR) mutant cells are defective in the proteolysis of abnormal proteins such as canavanine-containing proteins, puromycyl peptides, and the X90 fragment of ,B-galactosidase (2,11). This decrease occurs even in rpoH lon mutant bacteria, suggesting that other members of the heat shock regulon are also involved. Indeed, it has been observed that mutations in some of the other heat shock genes result in decreased proteolysis: dnaJ mutants are defective in the degradation of puromycyl peptides and a fragment of 3-galactosidase, groEL mutants are defective in the proteolysis of puromycyl peptides, and dnaK mutants are defective in the degradation of canavanine-containing proteins and puromycyl peptides (16, 25).Recently, the existence of a second heat shock regulon whose transcription is induced at a high temperature independently of rpoH (htpR) by a newly discovered sigma factor, Cr24 ((rE) was demonstrated (9,10,18,27 proteins (23, 24). This finding suggests that the HtrA (DegP) protein may itself be a protease or control the activit...

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