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Biomedical subjects

A Varshavsky

Publications and source records attributed to A Varshavsky.

At least 73 records · Page 4Linked to original sources
PubMed1990

cis-trans recognition and subunit-specific degradation of short-lived proteins.

The N-end rule, a code that relates the metabolic stability of a protein to the identity of its amino-terminal residue, is universal in that different versions of the N-end rule operate in mammals, yeast and bacteria (unpublished data). The N-end rule-based degradation signal comprises a destabilizing amino-terminal residue and a specific internal lysine residue. We now show that, in a multisubunit protein, these two determinants can be located on different subunits and still target the protein for destruction. Moreover, in this case (trans recognition) only the subunit that bears the lysine determinant is actually degraded. Thus an oligomeric protein can contain both short-lived and long-lived subunits. These insights have functional and practical implications.

Amino Acid Sequence↗
PubMed1990

In vivo degradation of a transcriptional regulator: the yeast alpha 2 repressor.

Metabolic instability is characteristic of regulatory proteins whose in vivo concentrations must vary as a function of time. The cell type-specific alpha 2 repressor of the yeast S. cerevisiae is shown here to have a half-life of only approximately 5 min. Each of the two structural domains of alpha 2 carries a sequence that can independently target a normally long-lived protein for rapid destruction. Moreover, these two degradation signals are shown to operate via distinct mechanisms. Mutants deficient in the degradation of alpha 2 have been isolated and found to have a number of additional defects, indicating that the pathways responsible for alpha 2 turnover include components with multiple functions. Finally, we demonstrate that a short-lived subunit of an oligomeric protein can be degraded in vivo without destabilizing other, long-lived subunits of the same protein. This subunit-specific degradation makes possible a novel type of posttranslational remodeling in which a heteromeric protein could be functionally modified by selective, degradation-mediated replacement of its subunits.

Amino Acid Sequence↗
PubMed1990

Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae.

Aminoacyl-tRNA-protein transferases (Arg-transferases) catalyze post-translational conjugation of specific amino acids to the amino termini of acceptor proteins. A function of these enzymes in eukaryotes has been shown to involve the conjugation of destabilizing amino acids to the amino termini of short-lived proteins, these reactions being a part of the N-end rule pathway of protein degradation (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). We have cloned the ATE1 gene of the yeast Saccharomyces cerevisiae which encodes arginyl-tRNA-protein transferase. ATE1 gives rise to a approximately 1.6-kilobase mRNA and codes for a 503-residue protein. Expression of the yeast ATE1 gene in Escherichia coli, which lacks Arg-transferases, was used to show that the ATE1 protein possesses the Arg-transferase activity. Null ate1 mutants are viable but lack the Arg-transferase activity and are unable to degrade those substrates of the N-end rule pathway that start with residues recognized by the Arg-transferase.

Acyltransferases↗
PubMed1990

The recognition component of the N-end rule pathway.

The N-end rule-based degradation signal, which targets a protein for ubiquitin-dependent proteolysis, comprises a destabilizing amino-terminal residue and a specific internal lysine residue. We report the isolation and functional analysis of a gene (UBR1) for the N-end recognizing protein of the yeast Saccharomyces cerevisiae. UBR1 encodes a approximately 225 kd protein with no significant sequence similarities to other known proteins. Null ubr1 mutants are viable but are unable to degrade the substrates of the N-end rule pathway. These mutants are partially defective in sporulation and grow slightly more slowly than their wild-type counterparts. The UBR1 protein specifically binds in vitro to proteins bearing amino-terminal residues that are destabilizing according to the N-end rule, but does not bind to otherwise identical proteins bearing stabilizing amino-terminal residues.

Amino Acid Sequence↗
PubMed1989

Universality and structure of the N-end rule.

Our previous work has shown that, in the yeast Saccharomyces cerevisiae, any of the eight stabilizing amino-terminal residues confers a long (greater than 20 h) half-life on a test protein beta-galactosidase (beta gal), whereas 12 destabilizing amino-terminal residues confer on beta gal half-lives from less than 3 min to 30 min. We now show that an analogous single-residue code (the N-end rule) operates in an in vitro system derived from mammalian reticulocytes. We also show that the N-end rule has a hierarchical structure. Specifically, amino-terminal Glu and Asp (and also Cys in reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to primary destabilizing residues such as Arg. Amino-terminal Gln and Asn are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into secondary destabilizing residues Glu and Asp. Furthermore, in reticulocytes, distinct types of the N-end-recognizing activity are shown to be specific for three classes of primary destabilizing residues: basic (Arg, Lys, His), bulky hydrophobic (Phe, Leu, Trp, Tyr), and small uncharged (Ala, Ser, Thr). Features of the N-end rule in reticulocytes suggest that the exact form of the N-end rule may depend on the cell's physiological state, thereby providing a mechanism for selective destruction of preexisting proteins upon cell differentiation.

Amino Acid Sequence↗
PubMed1989

The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein.

Mammalian tumours displaying multidrug resistance overexpress a plasma membrane protein (P-glycoprotein), which is encoded by the MDR1 gene and apparently functions as an energy-dependent drug efflux pump. Tissue-specific expression of MDR1 and other members of the MDR gene family has been observed in normal cells, suggesting a role for P-glycoproteins in secretion. We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a protein very similar to mammalian P-glycoproteins. Deletion of this gene resulted in sterility of MATa, but not of MAT alpha cells. Subsequent analysis revealed that the yeast P-glycoprotein is the product of the STE6 gene, a locus previously shown to be required in MATa cells for production of a-factor pheromone. Our findings suggest that the STE6 protein functions to export the hydrophobic a-factor lipopeptide in a manner analogous to the efflux of hydrophobic cytotoxic drugs catalysed by the related mammalian P-glycoprotein. Thus, the evolutionarily conserved family of MDR-like genes, including the hlyB gene of Escherichia coli and the STE6 gene of S. cerevisiae, encodes components of secretory pathways distinct from the classical, signal sequence-dependent protein translocation system.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis.

Three of the four yeast ubiquitin genes encode hybrid proteins which are cleaved to yield ubiquitin and previously unidentified ribosomal proteins. The transient association between ubiquitin and these proteins promotes their incorporation into nascent ribosomes and is required for efficient ribosome biogenesis. These results suggest a novel 'chaperone' function for ubiquitin, in which its covalent association with other proteins promotes the formation of specific cellular structures.

Blotting, Northern↗
PubMed1989

The degradation signal in a short-lived protein.

Our previous work has shown that the amino-terminal residue of a short-lived protein is a distinct component of the protein's degradation signal. To define the complete signal, otherwise identical dihydrofolate reductase test proteins bearing different extensions and either a "stabilizing" or a "destabilizing" amino-terminal residue were expressed in the yeast S. cerevisiae and their in vivo half-lives compared. The amino-terminal degradation signal is shown to comprise two distinct determinants. One, discovered previously, is the protein's amino-terminal residue. The second determinant, identified in the present work, is a specific lysine residue whose function in the degradation signal is not dependent on the unique amino acid sequences in the vicinity of the residue. The mechanistic significance of the second determinant is illuminated by the finding that in a targeted, short-lived protein, a chain of branched ubiquitin-ubiquitin conjugates is confined to a lysine residue that has been identified in the present work as the second determinant of the degradation signal.

Amino Acid Sequence↗
PubMed1989

A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.

The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.

DNA Mutational Analysis↗
PubMed1989

A DNA binding protein that recognizes oligo(dA).oligo(dT) tracts.

Oligo(dA).oligo(dT) tracts are common intergenic sequences in many organisms. In the yeast Saccharomyces cerevisiae, these sequences have been shown to influence transcription of adjacent genes. We have purified an oligo(dA).oligo(dT)-binding protein from S. cerevisiae and cloned its gene. This protein, which has been named datin, requires at least 9-11 bp of oligo(dA).oligo(dT) DNA for high affinity binding. The gene for datin (the DAT gene) encodes a 248-residue protein which contains a number of repeated sequence motifs. Datin purified from yeast corresponds to the N-terminal half of the DAT gene product. Null mutants in the DAT gene are viable but phenotypically distinguishable from congenic wild-type strains. We discuss unusual structural features and biochemical properties of datin in relation to its possible functions.

Amino Acid Sequence↗
PubMed1988

The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme.

Mutants in the gene CDC34 of the yeast Saccharomyces cerevisiae are defective in the transition from G1 to the S phase of the cell cycle. This gene was cloned and shown to encode a 295-residue protein that has substantial sequence similarity to the product of the yeast RAD6 gene. The RAD6 gene is required for a variety of cellular functions including DNA repair and was recently shown to encode a ubiquitin-conjugating enzyme. When produced in Escherichia coli, the CDC34 gene product catalyzed the covalent attachment of ubiquitin to histones H2A and H2B in vitro, demonstrating that the CDC34 protein is another distinct member of the family of ubiquitin-conjugating enzymes. The cell cycle function of CDC34 is thus likely to be mediated by the ubiquitin-conjugating activity of its product.

Amino Acid Sequence↗
PubMed1988

Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene.

We have used formaldehyde-mediated protein-DNA crosslinking within intact cells to examine the in vivo chromatin structure of the D. melanogaster heat shock protein 70 (hsp70) genes. In agreement with previous in vitro studies, we find that the heat shock-mediated transcriptional induction of the hsp70 genes perturbs their chromatin structure, resulting in fewer protein-DNA contacts crosslinkable in vivo by formaldehyde. However, contrary to earlier in vitro evidence that histones may be absent from actively transcribed genes, we show directly, by immunoprecipitation of in vivo-crosslinked chromatin fragments, that at least histone H4 remains bound to hsp70 DNA in vivo, irrespective of its rate of transcription. The formaldehyde-based in vivo mapping techniques described in this work are generally applicable, and can be used both to probe protein-DNA interactions within specific genes and to determine the genomic location of specific chromosomal proteins.

Animals↗
PubMed1988

Hypersensitivity to heavy water: a new conditional phenotype.

Wild-type strains of the yeast S. cerevisiae can grow on media containing 90% D2O. Using chemical mutagenesis we obtained a number of strains that grow on H2O-containing media but not on otherwise identical media containing 90% D2O. The frequency of these D2O-sensitive (d) mutants is comparable to the frequency of conventional temperature-sensitive (ts) mutants in the same mutagenized sample, and the ds mutations are distributed over a large number of complementation groups. Furthermore, most ds mutants do not display other conditional phenotypes, such as heat, cold, or osmotic sensitivity. Conversely, of 17 cell division cycle ts mutants tested, only 2 are also ds. Thus, the ds technique should be useful for producing conditional mutations in genes that are not amenable to the ts and cs approaches, and also for generating alternative conditional (ds) alleles in many other genes. In addition, the ds technique should make it possible to generate conditional (ds) mutants in homeothermic animals, thereby extending the advantages of conditional phenotypes to mammalian and avian genetics.

Culture Media↗
PubMed1987

The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses.

Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in eukaryotes. In the yeast Saccharomyces cerevisiae, four distinct ubiquitin-coding loci have been described. UBI1, UBI2, and UBI3 each encode hybrid proteins in which ubiquitin is fused to unrelated sequences. The fourth gene, UBI4, contains five ubiquitin-coding elements in a head-to-tail arrangement, and thus encodes a polyubiquitin precursor protein. A precise, oligonucleotide-directed deletion of UBI4 was constructed in vitro and substituted in the yeast genome in place of the wild-type allele. ubi4 deletion mutants are viable as vegetative cells, grow at wild-type rates, and contain wild-type levels of free ubiquitin under exponential growth conditions. However, although ubi4/UBI4 diploids can form four initially viable spores, the two ubi4 spores within the ascus lose viability extremely rapidly, apparently a novel phenotype in yeast. Furthermore, ubi4/ubi4 diploids are sporulation-defective. ubi4 mutants are also hypersensitive to high temperatures, starvation, and amino acid analogs. These three conditions, while diverse in nature, are all known to induce stress proteins. Expression of the UBI4 gene is similarly induced by either heat stress or starvation. These results indicate that UBI4 is specifically required for the resistance of cells to stress, and that ubiquitin is an essential component of the stress response system.

Alleles↗
PubMed1987

The yeast ubiquitin genes: a family of natural gene fusions.

Ubiquitin is a 76-residue protein highly conserved among eukaryotes. Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in vivo. We describe a family of four ubiquitin-coding loci in the yeast Saccharomyces cerevisiae. UB11, UB12 and UB13 encode hybrid proteins in which ubiquitin is fused to unrelated ('tail') amino acid sequences. The ubiquitin coding elements of UB11 and UB12 are interrupted at identical positions by non-homologous introns. UB11 and UB12 encode identical 52-residue tails, whereas UB13 encodes a different 76-residue tail. The tail amino acid sequences are highly conserved between yeast and mammals. Each tail contains a putative metal-binding, nucleic acid-binding domain of the form Cys-X2-4-Cys-X2-15-Cys-X2-4-Cys, suggesting that these proteins may function by binding to DNA. The fourth gene, UB14, encodes a polyubiquitin precursor protein containing five ubiquitin repeats in a head-to-tail, spacerless arrangement. All four ubiquitin genes are expressed in exponentially growing cells, while in stationary-phase cells the expression of UB11 and UB12 is repressed. The UB14 gene, which is strongly inducible by starvation, high temperatures and other stresses, contains in its upstream region strong homologies to the consensus 'heat shock box' nucleotide sequence. Elsewhere we show that the essential function of the UB14 gene is to provide ubiquitin to cells under stress.

Amino Acid Sequence↗