Milton J. Schlesinger

Uncoating ATPase is a member of the 70 kilodalton family of stress proteins

The synthetic peptide, VGIDLGTTYSC, derived from the heat shock-induced genes human hsp70, Drosophila hsp70, S. cerevisiae YG100, and E. coli dnaK, elicited antibodies that recognized two constitutive proteins in bovine extracts. One of these proteins, 71 kd, has previously been identified as uncoating ATPase, an enzyme that releases clathrin from coated vesicles. This immunological data complemented the result that uncoating ATPase was indistinguishable from the constitutive mammalian 71 kd stress protein by either partial proteolytic mapping or two-dimensional gel analysis. In addition, affinity-purified uncoating ATPase antibodies recognize proteins in yeast identified as the gene products of the heat shock or heat shock cognate genes YG100 and YG102. The results show that uncoating ATPase is a member of the 70 kd heat shock protein family.

The Heat Shock Respons

The response of cells to a heat shock or other stresses is the activation of a small number of genes which were previously inactive or transcribed at low levels. This response has been observed in a wide variety of bacterial, plant, and animal species. Evidence is accumulating that at least some of the proteins found in diverse species are similar, indicating a conservation of the response and the proteins in evolution. In a number of organisms a strong positive correlation has been found between the presence of heat shock proteins and ability of the organism to withstand thermal stress. This review attempts to assess the available data concerning the homology of proteins in different species, the localization of the proteins in cells, and the relationship between heat shock proteins and thermoresistance.

Ubiquitin is a heat shock protein in chicken embryo fibroblasts.

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.

Heat Shock RNA Levels in Brain and Other Tissues After Hyperthermia and Transient Ischemia

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70‐kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of ± the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several‐fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70‐related cDNA probes. Transient ischemia resulted in 5‐ to 10‐fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia. These studies emphasize the importance of using probes selective for stress‐inducible hsp70 sequences in evaluation of the heat shock response, particularly in tissues such as brain in which there is significant constitutive expression of hsp70‐related proteins.

Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein

Abstract The glycoprotein (G) of vesicular stomatitis virus (VSV) binds 1–2 moles of fatty acid per mole of protein. The fatty acids cannot be released by repeated extractions of the protein with organic solvents, nor can they be released by denaturing the protein with ionic or nonionic detergents. Pronase digestion of G yields an organic extractable fragment that contains bound fatty acid. The fatty acid is quantitatively released from this fragment and from intact G by mild alkali treatment in methanol and is identified by gas-liquid and thin-layer chromatography as, predominantly, the methyl ester of palmitic acid. Insignificant amounts of phosphate are found in G, thus ruling out the presence of bound phospholipid. Chicken embryo fibroblast pre-labeled with 3 H-palmitate and then infected with VSV for 4 hr show the presence of 3 H label in G but not in other viral structural proteins. The 3 H label is present only in the fatty acid moiety of the protein. Much smaller amounts of 3 H fatty acid are bound to G protein formed by the VSV mutant ts045 grown at the nonpermissive temperature, and no 3 H fatty acid is bound to G synthesized at 37°C in cells pretreated with tunicamycin, an inhibitor of glycosylation. However, infection with the VSV-Orsay strain at 30°C in the presence of tunicamycin allows for production of VSV particles with nonglycosylated G (Gibson, Schlesinger and Kornfeld, 1979), and this G has the same proportion of the fatty acid as does the normal glycosylated G. These data indicate that fatty acids become covalently attached to the G polypeptide chain during maturation of the protein—perhaps as the glycoprotein moves to the cells plasma membrane.

Vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores.

Abstract The effect of two cationic ionophores, monensin and A23187, on the replication of Sindbis and vesicular stomatitis virus in cultures of BHK and chicken embryo fibroblast cells has been studied. Treating these cells with the ionophores at 10 −6 , M 2 hr prior to infection at high multiplicities of infection did not affect viral protein synthesis but severely inhibited release of viral particles into the media. Glycoproteins of these viruses were synthesized in normal amounts but they did not appear on the surface of infected cells. Proteolytic cleavage of Sindbis virus glycoprotein PE2 to E2 was inhibited in ionophore-treated cells, although fatty acid attachment to PE2 proceeded normally. Fatty acid attachment to VSV G protein and the posttranslational removal of mannose residues from G oligosaccharides occurred in the drug-treated cells. These data indicate that these ionophores block the movement of viral glycoproteins from the Golgi apparatus to the cell surface membrane where budding and release of these two viruses occur.

How the cell copes with stress and the function of heat shock proteins.

ABSTRACT: Virtually all cells, including the prokaryotic microorganisms and the highly differentiated eukaryotic cells in human tissues, contain a small set of normally silent genes that are rapidly activated by a heat shock that raises the temperature only 5 to 10% above that of the normal physiologic range for that organism. Concomitantly, many active genes are turned off. Other kinds of stress, such as exposure to alcohol or other organic agents, heavy metals, oxidants, and agents capable of perturbing protein structure, produce a similar response, and many of these activate the same set of genes. The proteins encoded by these stress-activated genes are called heat shock proteins (hsp). They are strongly conserved in structure among widely divergent biologic species, and many function as “molecular chaperones” by forming transient complexes with partially folded or misfolded polypeptides so as to prevent their irreversible denaturation. Most hsp are members of gene/protein families, and isoforms are frequently found under normal physiologic conditions in many compartments of the cell where they act also as chaperones, binding to a variety of polypeptides to facilitate folding, oligomerization, transport, metabolic activity, and degradation. Few of the polypeptide “targets” that complex with stress-induced forms of hsp have been identified, but a number of cellular components have been shown to be particularly stress sensitive. They include macromolecular complexes involved in the maintenance of chromosome replication and transcription, mRNA splicing, and ribosome assembly. Mitochondria and the intermediate filament network are also highly sensitive, whereas the protein synthetic machinery and vesicles of the secretory pathway are relative stable to physiologic stress. The factors regulating heat shock genes in the eukaryote are highly conserved among widely divergent species and include promoters consisting of arrays of short, inverted sequences in the DNA, called the heat shock element, and heat shock factors, which are large polypeptides that occupy these promoters soon after the cell senses the temperature shift. The sensor(s) that signal the cell to initiate binding of heat shock factors to the heat shock element, thereby activating gene transcription, have not been identified, but misfolded proteins are postulated to play a key role in this event. The response is also transient, and other factors down-regulate the system. Cells that have been mildly prestressed so that they contain significant levels of the hsp become tolerant to stress conditions that would normally kill the cell. In this way, organisms survive environmental conditions that might otherwise prove fatal.

The Sindbis virus 6K protein can be detected in virions and is acylated with fatty acids.

A small hydrophobic polypeptide is encoded within the genome of the alphaviruses by a set of 165 nucleotides which map between the sequences for the two virus glycoproteins. This polypeptide has been referred to as 6K and was previously found on membranes in virus-infected cells. We report here that this protein is heavily acylated with long chain fatty acids covalently attached in hydroxylamine-sensitive ester bonds and that the 6K protein can be detected in purified preparations of virions. A polyclonal rabbit serum, raised against a peptide which contained the 16 amino acids at the amino-terminus of the 6K protein, was used to identify the 6K protein in infected cells and virions. This antibody also precipitated a 4K protein which was present in Sindbis virus-infected cells but not in virions. This latter protein was shown to be an underacylated form of the 6K protein and infected cells contained about twice as much 4K as 6K. In the cell there was close to a 1:1 stoichiometry between the 4K + 6K proteins and the virus glycoproteins E1, p62, and E2, but in virions the ratio of 6K to E1 + E2 ranged from 0.08 to 0.12.

Evidence for an autoprotease activity of sindbis virus capsid protein.

Abstract Sindbis virus capsid protein is virtually the only product formed when viral 26 S RNA is added to a mouse Krebs ascites cell-free protein synthesis system. However, substitution of arginine and proline by the respective analogues canavanine and azetidine-2-carboxylate inhibits capsid production and larger polypeptides accumulate. The latter are converted to capsid in pulse-chase experiments when the normal amino acids are added during the chase, but not if the chase period contains only the analogues in the reaction mixture. These results support an autoprotease model for the co-translational cleavage of Sindbis virus capsid proteins.

Site-directed mutations in sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding

Site-directed mutagenesis was used to obtain four mutants with amino acid replacements in the cytoplasmic domain of the E2 glycoprotein and three with replacements in the 6K protein of Sindbis virus. All but one of these mutants yielded progeny virus after transfection of chicken embryo fibroblasts with RNA prepared by in vitro transcription of the virus cDNA; however, even this nonproducer mutant made virus structural proteins in the transfected cells. The other six mutants divided into two groups based on growth in chicken embryo fibroblasts. One group of four mutants (two in E2 and two in 6K) was indistinguishable from wild-type in formation of infectious virus in avian cells while the other group, consisting of two mutants, grew significantly slower. All six mutants grew slower than the parental wild-type virus in mosquito cells. In avian cells, all mutants produced extracellular particles at a slower rate than the wild-type and many of the particles contained multiple nucleocapsids, based on electron microscopy and kinetics of thermal inactivation. One of the E2 mutants with a cysteine changed to alanine and the 6K mutant with four cysteines replaced were deficient in covalent-bound palmitic acid. Two mutants with changes near the signalase cleavage sites between E2 and 6K and between 6K and E1 appeared to be defective in proteolytic processing. Despite individual differences, all of these mutants and the two previously described produced similar phenotypes in which multicored infectious virus particles were released more slowly from mosquito cells than from avian cells.