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H R Pelham

Publications and source records attributed to H R Pelham.

At least 91 records · Page 5Linked to original sources
PubMed1988

Sorting of soluble ER proteins in yeast.

In animal cells, luminal endoplasmic reticulum (ER) proteins are prevented from being secreted by a sorting system that recognizes the C-terminal sequence KDEL. We show that yeast has a similar sorting system, but it recognizes HDEL, rather than KDEL: derivatives of the enzyme invertase that bear the HDEL signal fail to be secreted. An invertase fusion protein that is retained in the cells is partially modified by outer-chain mannosyl transferases, which reside in the Golgi element. This supports the view, based on studies in animal cells, that ER targeting is achieved by continuous retrieval of proteins from the Golgi. We have used an invertase fusion gene to screen for mutants that are defective in this sorting system. Over 60 mutants were obtained; eight of these are alleles of a single gene, erd1. The mutant strains grow normally at 30 degrees C, but instead of retaining the fusion protein in the cells, they secrete it.

Amino Acid Sequence↗
PubMed1988

Seventy-kilodalton heat shock proteins and an additional component from reticulocyte lysate stimulate import of M13 procoat protein into microsomes.

Processing of M13 procoat protein, synthesized in a bacterial cell-free extract, to transmembrane coat protein by dog pancreas microsomes is stimulated by a system which is present in rabbit reticulocytes and depends on nucleoside triphosphates. This system consists of (at least) two components which act synergistically: members of the 70-kd heat shock protein family and (at least) one additional component. This component depends on ATP (or GTP) for its action.

Adenosine Triphosphate↗
PubMed1988

Constitutive binding of yeast heat shock factor to DNA in vivo.

We measured the binding of yeast heat shock factor (HSF) to DNA in vivo by using an interference assay in which HSF excludes GAL4 from a synthetic promoter element containing overlapping binding sites for each protein. The results show that HSF binds to DNA in unstressed cells and that binding is not sufficient for transcriptional activation.

Base Sequence↗
PubMed1987

An ecdysone response element in the Drosophila hsp27 promoter.

It has previously been shown that a region of 100 bp in the Drosophila hsp27 promoter is sufficient to confer ecdysone inducibility on a heterologous gene. We now show, using binding and DNase I footprinting assays, that a 23-bp hyphenated dyad within this sequence forms a protein-binding site, and that this is sufficient for inducibility. The sequence shows partial homology with mammalian steroid receptor binding sites. UV crosslinking identifies an 80- to 90-kd protein that binds specifically to this sequence and is thus a candidate for the ecdysone receptor.

Journal Article↗
PubMed1987

The glucose-regulated protein grp94 is related to heat shock protein hsp90.

We report the sequence of a cDNA clone that encodes the C-terminal half of the hamster 94 X 10(3) Mr glucose-regulated protein, grp94. The amino acid sequence of this protein is about 50% homologous to Drosophila hsp83 and yeast hsp90, suggesting that grp94 and hsp90 have similar functional properties. Unlike hsp90, grp94 is associated with the endoplasmic reticulum. It has the same C-terminal tetrapeptide as two other luminal endoplasmic reticulum proteins, grp78 and protein disulphide isomerase. We suggest that this sequence forms part of a signal for retention of proteins in the lumen of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1987

A C-terminal signal prevents secretion of luminal ER proteins.

Proteins that permanently reside in the lumen of the endoplasmic reticulum (ER) must somehow be distinguished from newly synthesized secretory proteins, which pass through this compartment on their way out of the cell. Three luminal ER proteins whose sequence is known, grp78 ("BiP"), grp94, and protein disulphide isomerase, share the carboxy-terminal sequence Lys-Asp-Glu-Leu (KDEL). We show that deletion (or extension) of the carboxyl terminus of grp78 results in secretion of this protein when it is expressed in COS cells. Conversely, a derivative of chicken lysozyme containing the last six amino acids of grp78 fails to be secreted and instead accumulates in the ER. We propose that the KDEL sequence marks proteins that are to be retained in the ER and discuss possible retention mechanisms.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of a heat-shock element binding protein from yeast.

The promoters of heat shock genes are activated when cells are stressed. Activation is dependent on a specific DNA sequence, the heat-shock element (HSE). We describe the purification to homogeneity of an HSE-binding protein from yeast (Saccharomyces cerevisiae), using sequential chromatography of whole cell extracts on heparin-agarose, calf thymus DNA-Sepharose and an affinity column consisting of a repetitive synthetic HSE sequence coupled to Sepharose. The protein runs as a closely spaced doublet of approximately 150 kd on SDS-polyacrylamide gels; mild proteolysis generates a stable 70-kd fragment which retains DNA binding activity. The relative affinities of the protein for a range of variant HSE sequences correlates with the ability of these sequences to support heat-inducible transcription in vivo, suggesting that this polypeptide is involved in the activation of heat-shock promoters. However, the protein was purified from unshocked yeast, and may therefore represent an unactivated form of heat-shock transcription factor. Study of the purified protein should help to define the mechanistic basis of the heat-shock response.

Base Sequence↗
PubMed1987

Cloning and expression of a gene encoding hsc73, the major hsp70-like protein in unstressed rat cells.

The isolation and complete DNA sequence of a rat genomic clone encoding hsc73, the major hsp70-like protein found in growing cells is described. Unlike the heat-inducible genes characterized so far, the hsc73 gene is interrupted by introns, and there are also numerous intronless hsc73 pseudogenes in the rat genome. We show that the levels of hsc73 mRNA are approximately 5-fold higher in rapidly growing tissue-culture cells than in cells whose growth has been arrested by serum starvation. The abundance of hsc73 mRNA is not significantly increased by heat shock of either fed or starved cells. The hsc73 promoter contains putative binding sites for transcription factor Sp1, two CCAAT boxes and, surprisingly, two matches to the consensus heat-shock regulatory element. When fused to the CAT gene and transfected into COS or HeLa cells, the promoter is constitutively active, showing only a small induction by heat shock. Deletion of some constitutive elements makes it more strongly heat-inducible. The gene thus appears to be subject to more than one form of regulation, mediated by different promoter elements that are intermingled.

Amino Acid Sequence↗
PubMed1986

An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein.

We have characterized a cDNA clone that encodes a protein related to the 70 kd heat shock protein, but is expressed in normal rat liver. This protein has a hydrophobic leader and is secreted into the endoplasmic reticulum. We show that it is identical with two previously described proteins: GRP78, whose synthesis is induced by glucose starvation, and BiP, which is found bound to immunoglobulin heavy chains in pre-B cells. This protein, which is abundant in antibody-secreting cells, can be released from heavy chains by ATP, a reaction analogous to the release of hsp70 from heat shocked nuclear structures. We propose a specific role for this protein in the assembly of secreted and membrane-bound proteins.

Adenosine Triphosphate↗
PubMed1986

Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter.

The Xenopus hsp70 promoter contains three copies of the consensus heat shock element (HSE) between positions -260 and -100. When the gene is transfected into mammalian cells, maximal heat-induced expression requires two HSEs in addition to a CCAAT box located next to the TATA box. The HSE-containing region can be separated from the CCAAT/TATA region without affecting expression of the gene, and it can enhance transcription of a linked beta-globin gene upon heat shock. It thus has the properties of a heat-inducible enhancer. Such an enhancer can also be generated by duplication of HSE sequences from the Drosophila hsp70 promoter, which were previously identified as an upstream promoter element and are known to bind a purified heat shock transcription factor in vitro.

Animals↗
PubMed1986

Activation of the Drosophila hsp27 promoter by heat shock and by ecdysone involves independent and remote regulatory sequences.

Transcription of the Drosophila hsp27 gene is induced both by heat shock and by the steroid hormone ecdysone. Deletion analysis of the promoter using a transient expression assay in Drosophila tissue culture cells reveals two separate and functionally independent regulatory regions, whose positions coincide with previously reported DNase I-hypersensitive sites in embryonic chromatin. Ecdysone induction is mediated by multiple elements between -579 and -455. Several matches to the consensus heat shock regulatory element (HSE) lie between -370 and -270, and deletion of these abolishes the response to heat shock. Heat inducibility can be restored by the addition of HSE sequences from the hsp70 promoter, even when these are placed at the 3' end of the gene. Furthermore, HSEs seperated by >2 kb can interact in a cooperative way to promote transcription. Deletion of sequences adjacent to the TATA box (-40 to -154) has little effect on induction by either heat shock or ecdysone. In this respect, activation of the hsp27 TATA box in Drosophila cells differs from the long-range activation of promoters by HSEs or viral enhancers in HeLa cells.

Journal Article↗
PubMed1985

Involvement of ATP in the nuclear and nucleolar functions of the 70 kd heat shock protein.

The major heat shock protein, hsp70, is an ATP-binding protein which is synthesized in very large amounts in response to stress. In unstressed, or recovered, mammalian cells it is found in both nucleus and cytoplasm. Under these conditions, its interaction with nuclei is weak, and it is readily released from them upon lysis of cells in isotonic buffer. After heat shock, hsp70 binds tightly first to some nuclear component(s) and then to nucleoli. It can be released from these binding sites rapidly and specifically in vitro by as little as 1 microM ATP, but not by non-hydrolysable ATP analogues. Studies of hsp70 deletion mutations show that the ability of mutants to be released by ATP correlates with their ability to migrate to heat-shocked nucleoli and aid their repair in vivo. We propose a model in which ATP-driven cycles of binding and release of hsp70 help to solubilize aggregates of proteins or RNPs that form after heat shock. Cells also contain proteins related to hsp70 that are synthesized in the absence of stress. The most abundant of these shows the same behaviour as hsp70 after heat shock, and thus may perform a related function in both normal and stressed cells.

Adenosine Triphosphate↗
PubMed1985

Transcription of Drosophila small hsp-tk hybrid genes is induced by heat shock and by ecdysterone in transfected Drosophila cells.

Hybrid genes containing the 5' region of Drosophila heat shock protein (hsp) genes, ligated to the herpesvirus thymidine kinase (tk) gene, were transfected into Drosophila line S3 cells. Constructions containing sequences upstream from hsp70, or from any of the small hsp genes, showed heat-inducible tk expression. Ecdysterone-inducible tk expression was seen only in transfections with small hsp-tk hybrid genes. Plasmids containing a synthetic "heat shock consensus sequence" or a deletion of the hsp23 upstream region were totally inactive in transfection studies.

Animals↗
PubMed1984

Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70.

We have developed a technique which allows specific detection of proteins expressed from cloned genes. The method involves fusion of an oligonucleotide coding for part of the neuropeptide substance P to the 3' end of the gene; the protein can then be detected with a monoclonal antibody that recognises this peptide. We have used this method to determine the properties of deletion mutants of the major Drosophila heat shock protein, hsp70, expressed in monkey COS cells. The results suggest that this protein has two distinct domains. Both are capable of accumulating in the nucleus of unstressed cells, but only the more highly conserved N-terminal domain is able to bind to nucleoli following a heat shock. This implies that nucleolar binding and nuclear migration are distinct properties of the protein, and suggests that the former may be of functional importance. In addition, we observed a novel effect of heat shock on cellular metabolism: protein fragments that are normally rapidly degraded are stabilized. The effect persists for several hours after the heat shock, but does not require expression of heat shock proteins. Together with previously published data, these results suggest an intimate relationship between protein degradation and the heat shock response.

Animals↗
PubMed1984

Hsp70 accelerates the recovery of nucleolar morphology after heat shock.

The major heat-shock protein, hsp70, is synthesized by cells of many organisms in response to stress. In the present study, Drosophila hsp70 was expressed from cloned genes in mouse L cells and monkey COS cells and detected by immunofluorescence using monoclonal antibodies. Hsp70 is found mostly but not exclusively in the nucleus of unstressed cells. For several hours after a short heat shock, however, it is strongly concentrated in nucleoli. Nucleoli are transiently damaged by such a heat shock: their morphology changes and assembly and export of ribosomes is blocked for several hours. This block can be visualized by addition of actinomycin D: under normal conditions pre-ribosomes are chased out of nucleoli, and the latter shrink dramatically, but no such shrinking is seen in heat-shocked cells. High levels of hsp70 can be produced in unstressed COS cells by transfecting them with an appropriate expression plasmid. Such cells show a more rapid recovery of nucleolar morphology following a heat shock than do untransfected cells. Furthermore, heat shock does not prevent shrinkage of their nucleoli in the presence of actinomycin, which indicates that ribosome export also recovers rapidly when pre-synthesized hsp70 is present. I suggest that an important function of hsp70 is to catalyze reassembly of damaged pre-ribosomes and other RNPs after heat shock.

Animals↗
PubMed1984

The use of promoter fusions in Drosophila genetics: isolation of mutations affecting the heat shock response.

We have constructed a gene fusion using the promoter of Drosophila hsp70 and the structural gene for Drosophila alcohol dehydrogenase (Adh) and used this construct to transform Adh-deficient flies. In these transformants, Adh is expressed only after heat shock. Like hsp70 itself, this heat-shock-inducible Adh (Adhhs) is induced in a wide variety of tissues. It fails to be induced in primary spermatocytes. Although the tissue distribution of Adh activity is very different from wild type, this does not appear to be deleterious. Indeed, the induction of Adhhs allows flies to survive exposure to ethanol. We have used this latter characteristic to select dominant, trans-acting mutations that alter the response of flies to heat shock.

Alcohol Oxidoreductases↗