Chaperones: helpers along the pathways to protein folding.
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Chaperonin 60↗
Biomedical subjects
E A Craig
Publications and source records attributed to E A Craig.
A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins.
The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix-targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt-hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt-hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.
Amino Acid Sequence↗
Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix.
To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.
Biological Transport↗
Ophthalmic disease in patients with unrecognized HIV infection.
HIV disease has a long and relatively asymptomatic incubation period before the onset of AIDS. The ophthalmic manifestations of AIDS are well recognized. Little is known of the incidence or range of ophthalmic disease seen in the latent period of HIV disease. A retrospective analysis of 125 case notes of HIV 1 antibody positive patients was undertaken. Of these, 15 (12%) had previously attended the ophthalmic department, at which stage their HIV status was unknown. Eight patients were diagnosed as being HIV positive within twelve months of attending the ophthalmic department. There was a predominance of ophthalmic infections, as opposed to trauma which is the commonest cause for ophthalmic casualty referral. Some of the ophthalmic presentations seen were the initial presenting sign of HIV disease. There were also cases of serious trauma and a mixture of other ophthalmic diseases. We discuss these ophthalmic findings and their relevance to HIV disease.
Acquired Immunodeficiency Syndrome↗
Genetic evidence for a functional relationship between Hsp104 and Hsp70.
The phenotypes of single Hsp104 and Hsp70 mutants of the budding yeast Saccharomyces cerevisiae provide no clue that these proteins are functionally related. Mutation of the HSP104 gene severely reduces the ability of cells to survive short exposures to extreme temperatures (thermotolerance) but has no effect on growth rates. On the other hand, mutations in the genes that encode Hsp70 proteins have significant effects on growth rates but do not reduce thermotolerance. The absence of a thermotolerance defect in S. cerevisiae Hsp70 mutants is puzzling, since the protein clearly plays an important role in thermotolerance in a variety of other organisms. In this report, examination of the phenotypes of combined Hsp104 and Hsp70 mutants uncovers similarities in the functions of Hsp104 and Hsp70 not previously apparent. In the absence of the Hsp104 protein, Hsp70 is very important for thermotolerance in S. cerevisiae, particularly at very early times after a temperature upshift. Similarly, Hsp104 plays a substantial role in vegetative growth under conditions of decreased Hsp70 protein levels. These results suggest a close functional relationship between Hsp104 and Hsp70.
DNA Mutational Analysis↗
Saccharomyces cerevisiae HSP70 heat shock elements are functionally distinct.
The Saccharomyces cerevisiae HSP70 gene SSA1 has multiple heat shock elements (HSEs). To determine the significance of each of these sequences for expression of SSA1, we analyzed expression from a set of promoters containing point mutations in each of the HSEs, individually and in pairwise combinations. Of the three HSE-like sequences, two (HSE2 and HSE3) were active promoter elements; only one, HSE2, was active under basal growth conditions. Either HSE2 or HSE3 alone was able to drive SSA1 transcription at near-normal rates after heat shock. Both HSE2 and HSE3 were capable of driving basal transcription when placed in the context of the CYC1 promoter. Previous analysis had identified an upstream repressing sequence overlapping HSE2 that repressed basal transcription driven by HSE2. Our analysis showed that basal transcription driven by HSE3 was repressed both by the distant upstream repressing sequence and by closer flanking sequences. The ability to drive basal transcription is not inherent in all natural HSEs, since the HSEs from the heat-inducible SSA3 and SSA4 genes showed no basal activity when placed in the CYC1 vector. Gel mobility shift experiments showed that the same population of heat shock transcription factor molecules bound to HSEs capable of driving basal activity and to HSEs having very low or undetectable basal activity.
Base Sequence↗
Heat shock proteins: molecular chaperones of protein biogenesis.
Heat shock proteins (Hsps) were first identified as proteins whose synthesis was enhanced by stresses such as an increase in temperature. Recently, several of the major Hsps have been shown to be intimately involved in protein biogenesis through a direct interaction with a wide variety of proteins. As a reflection of this role, these Hsps have been referred to as molecular chaperones. Hsp70s interact with incompletely folded proteins, such as nascent chains on ribosomes and proteins in the process of translocation from the cytosol into mitochondria and the endoplasmic reticulum. Hsp60 also binds to unfolded proteins, preventing aggregation and facilitating protein folding. Although less well defined, other Hsps such as Hsp90 also play important roles in modulating the activity of a number of proteins. The function of the proteolytic system is intertwined with that of molecular chaperones. Several components of this system, encoded by heat-inducible genes, are responsible for the degradation of abnormal or misfolded proteins. The budding yeast Saccharomyces cerevisiae has proven very useful in the analysis of the role of molecular chaperones in protein maturation, translocation, and degradation. In this review, results of experiments are discussed within the context of experiments with other organisms in an attempt to describe the current state of understanding of these ubiquitous and important proteins.
Cytosol↗
Multiple protein tyrosine phosphatase-encoding genes in the yeast Saccharomyces cerevisiae.
In higher eukaryotic organisms, the regulation of tyrosine phosphorylation is known to play a major role in the control of cell division. Recently, a wide variety of protein tyrosine phosphatase (PTPase)-encoding genes (PTPs) have been identified to accompany the many tyrosine kinases previously studied. However, in the yeasts, where the cell cycle has been most extensively studied, identification of the genes involved in the direct regulation of tyrosine phosphorylation has been difficult. We have identified a pair of genes in the yeast Saccharomyces cerevisiae, which we call PTP1 and PTP2, whose products are highly homologous to PTPases identified in other systems. Both genes are poorly expressed, and contain sequence elements consistent with low-abundance proteins. We have carried out an extensive genetic analysis of PTP1 and PTP2, and found that they are not essential either singly or in combination. Neither deletion nor overexpression results in any strong phenotypes in a number of assays. Deletions also do not affect the mitotic blockage caused by deletion of the MIH1 gene (encoding a positive regulator of mitosis) and induction of the heterologous Schizosaccharomyces pombe wee1+ gene (encoding a negative regulator of mitosis). Molecular analysis has shown that PTP1 and PTP2 are quite different structurally and are not especially well conserved at the amino acid sequence level. Low-stringency Southern blots indicate that yeast may contain a family of PTPase-encoding genes. These results suggest that yeast may contain other PTPase-encoding genes that overlap functionally with PTP1 and PTP2.
Amino Acid Sequence↗
The translation machinery and 70 kd heat shock protein cooperate in protein synthesis.
The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that Ssb1/2p may bind directly to the nascent polypeptide. Mutant ssb1 ssb2 strains grow slowly, contain a low number of translating ribosomes, and are hypersensitive to several inhibitors of protein synthesis. The slow growth phenotype of ssb1 ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor 1 alpha (EF-1 alpha)-like protein. We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptide chain through the ribosome in a manner analogous to the role played by organelle-localized hsp70 in the transport of proteins across membranes.
Amino Acid Sequence↗
TCP1 - molecular chaperonin of the cytoplasm?
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Journal Article↗
Isolation and characterization of extragenic suppressors of mutations in the SSA hsp70 genes of Saccharomyces cerevisiae.
Saccharomyces cerevisiae strains that contain null alleles of two hsp70 genes, SSA1 and SSA2, are temperature sensitive for growth. In this study, extragenic suppressors of ssa1 ssa2 have been isolated. Suppression is due to mutations at nuclear loci designated EXA1, EXA2 and EXA3 for EXtragenic suppressor hsp70 subfamily A. Two of the four EXA1 alleles are dominant as is EXA3-1. The other two EXA1 alleles as well as the sole EXA2 allele are recessive. EXA1 mutations lead to accumulation of a previously uncharacterized form of hsp70. EXA2 and EXA3 mutations affect the regulation of the stress response. In exa2-1 ssa1 ssa2 strains the gene products of the remaining SSA hsp70 genes, SSA3 and SSA4 (Ssa3/4p), accumulate to higher levels. The EXA3-1 mutation results in increased accumulation of both Ssa3/4p and the hsp70s encoded by the SSB1 and SSB2 genes (Ssb1/2p), suggesting that the EXA3 gene product plays a central role in the yeast stress response. Consistent with this hypothesis, EXA3-1 is tightly linked to HSF1, the gene encoding the transcriptional regulatory protein known as "heat shock factor." All of the genes identified in this study seem to be involved in regulating the expression of SSA3 and SSA4 or the activity of their protein products.
Alleles↗
Facial reconstruction.
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Anthropology, Physical↗
Uncoating of coated vesicles by yeast hsp70 proteins.
The ability of hsp70 isoenzymes from wild-type and mutant yeast strains to uncoat bovine brain coated vesicles was analyzed and compared with that of the brain uncoating ATPase. Results show that, among the four major cytoplasmic isoenzymes produced in wild-type yeast, almost all of the activity is associated with the SSA1 and SSA2 isoenzymes. The SSB1 and SSB2 isoenzymes have almost no uncoating activity and are not found in the clathrin-hsp70 complexes formed during the uncoating reaction. Using hsp70 mutant yeast strains we find a marked difference in uncoating activity between the SSA1 and SSA2 isoenzymes, although there is only a 3% difference between their amino acid sequences. The SSA4 isoenzyme, which is produced only under stress conditions, has an uncoating activity intermediate between SSA1 and SSA2. These results suggest that the ability of hsp70 isoenzymes to uncoat clathrin-coated vesicles is restricted to certain members of the hsp70 family and can be affected by subtle changes in amino acid sequence. We also investigated the uncoating activity of mixtures of isoenzymes and find that the isoenzyme with lower uncoating activity reduces the activity of the isoenzyme with higher uncoating activity possibly by occupying binding sites on coated vesicles.
Adenosine Triphosphatases↗
Functional analysis of a conserved amino-terminal region of HSP70 by site-directed mutagenesis.
Hsp70 proteins have been highly conserved throughout evolution. As a first step in a structure-function analysis of hsp70, we constructed and analysed the consequences of mutations in a portion of the SSA1 gene, a member of the Saccharomyces cerevisiae HSP70 multigene family, that encodes a nearly invariant region near the amino terminus. Analysis of strains expressing SSA1 proteins with alterations at positions 8, 11 and 15 showed that these conserved residues within this region are critical for normal functioning of the protein. SSA1 protein containing either of two changes at position 15 was able to slightly complement the inviability of an ssa1ssa2ssa4 strain, but was inactive in other complementation assays. The other mutant proteins tested were unable to complement any tested phenotype. Effective interallelic complementation of several phenotypes was observed when a mutant protein substituted at position 8 was expressed in the same cell with either of two proteins carrying substitutions at position 15, suggesting that hsp70 acts as a multimer. Evidence from previous studies suggests that hsp70 proteins engage in ATP-driven cycles of binding and release from peptides. The ability of the mutant proteins to bind ATP and a peptide was tested. The Ssa1p carrying a substitution at position 8, which inhibits growth of cells carrying wild-type SSA proteins, showed a defect in release from a peptide relative to wild type. Two mutations, one each at position 8 and 15, resulted in accumulation of phosphorylated isoforms which may be a normal, transient hsp70 intermediate.
Adenosine Triphosphate↗
Inducing and assaying heat-shock response in Saccharomyces cerevisiae.
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Autoradiography↗
Is hsp70 the cellular thermometer?
Cells respond to an increase in temperature by inducing the synthesis of the heat shock proteins, which are a small set of evolutionarily conserved proteins. We review the evidence leading us to suggest that the free pool of one of these proteins, hsp70, serves as a cellular thermometer that regulates the expression of all heat shock proteins.
Animals↗
Transcriptional regulation of a yeast HSP70 gene by heat shock factor and an upstream repression site-binding factor.
SSA1, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, displays a significant basal level of expression under optimal growth conditions. Although multiple sites related to the heat shock element (HSE) consensus sequences are present in the SSA1 promoter region, one of these, HSE2, plays a key role in basal expression. An upstream repression site (URS) located adjacent to HSE2 causes repression of basal expression but has little effect on heat-inducible expression of SSA1. A series of DNase I footprinting assays suggests that heat shock factor (HSF) and a URS-binding factor (URSF) can bind simultaneously to the adjacent binding sites HSE2 and URS under optimal growth conditions. URSF in extracts from heat-shocked cells does not bind (or binds very poorly) to the URS adjacent to HSE2. However, URSF in these extracts is able to bind the URS if the URS is separated from HSE2 or if the HSE is mutated such that HSF binding is abolished. These in vitro experiments are consistent with in vivo results showing that the URS is able to repress transcription driven by HSE2 both before and after heat shock if it is separated from HSE2. Our results are consistent with a model of repression in which URSF and HSF bind simultaneously to the adjacent binding sites URS and HSE2 prior to heat shock. After heat shock, however, binding of the two proteins to the adjacent sites is exclusive, perhaps due to modification of HSF known to occur upon heat shock. Because HSF binding predominates, repression by URSF is relieved upon heat shock.
Base Sequence↗
Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix.
We previously reported that hsp70 in the mitochondrial matrix (mt-hsp 70 = Ssclp) is required for import of precursor proteins destined for the matrix or intermembrane space. Here we show that mt-hsp70 is also needed for the import of mitochondrial inner membrane proteins. In particular, the precursor of ADP/ATP carrier that is known not to interact with hsp60 on its assembly pathway requires functional mt-hsp70 for import, suggesting a general role of mt-hsp70 in membrane translocation of precursors. Moreover, a precursor arrested in contact sites was specifically co-precipitated with antibodies directed against mt-hsp70. We conclude that mt-hsp70 is directly involved in the translocation of many, if not all, precursor proteins that are transported across the inner membrane.
Biological Transport↗