Heat-shock proteins of Drosophila are associated with nuclease-resistant, high-salt-resistant nuclear structures.

Proteins produced in cultured Drosophila cells during the heat-shock response (HSPs) were recently shown by autoradiography to be confined in large measure to the cell nucleus. We report here that nuclear HSPs are not associated with nucleosomes solubilizes by treatment with staphylococcal nuclease at low ionic strength nor are HSPs released by extraction with high salt, which solubilized most of the remaining histones and DNA. Possible functions of nuclear HSPs are discussed.

Detecting and measuring cotranslational protein degradation in vivo.

Nascent polypeptides emerging from the ribosome and not yet folded may at least transiently present degradation signals similar to those recognized by the ubiquitin system in misfolded proteins. The ubiquitin sandwich technique was used to detect and measure cotranslational protein degradation in living cells. More than 50 percent of nascent protein molecules bearing an amino-terminal degradation signal can be degraded cotranslationally, never reaching their mature size before their destruction by processive proteolysis. Thus, the folding of nascent proteins, including abnormal ones, may be in kinetic competition with pathways that target these proteins for degradation cotranslationally.

Degradation of G alpha by the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Overexpression of targeting components of the N-end rule pathway in Saccharomyces cerevisiae inhibited the growth of haploid but not diploid cells. This ploidy-dependent toxicity was shown to result from enhanced degradation of Gpa1, the alpha subunit (G alpha) of a heterotrimeric guanine nucleotide-binding protein (G protein) that regulates cell differentiation in response to mating pheromones. Sst2, a protein whose absence renders cells hypersensitive to pheromone, was essential for degradation of G alpha but not other N-end rule substrates, suggesting the involvement of an indirect, or trans-, targeting mechanism. G alpha degradation by the N-end rule pathway adds another regulatory dimension to the multitude of signaling functions mediated by G proteins.

A yeast protein similar to bacterial two-component regulators.

Many bacterial signaling pathways involve a two-component design. In these pathways, a sensor kinase, when activated by a signal, phosphorylates its own histidine, which then serves as a phosphoryl donor to an aspartate in a response regulator protein. The Sln1 protein of the yeast Saccharomyces cerevisiae has sequence similarities to both the histidine kinase and the response regulator proteins of bacteria. A missense mutation in SLN1 is lethal in the absence but not in the presence of the N-end rule pathway, a ubiquitin-dependent proteolytic system. The finding of SLN1 demonstrates that a mode of signal transduction similar to the bacterial two-component design operates in eukaryotes as well.

Heat-inducible degron: a method for constructing temperature-sensitive mutants.

A temperature-sensitive (ts) mutant retains the function of a gene at a low (permissive) temperature but not at a high (nonpermissive) temperature. Arg-DHFR, a dihydrofolate reductase bearing an amino-terminal (N-terminal) arginine, is long-lived in the yeast Saccharomyces cerevisiae, even though arginine is a destabilizing residue in the N-end rule of protein degradation. A ts derivative of Arg-DHFR was identified that is long-lived at 23 degrees C but rapidly degraded by the N-end rule pathway at 37 degrees C. Fusions of ts Arg-DHFR to either Ura3 or Cdc28 of S. cerevisiae confer ts phenotypes specific for these gene products. Thus, Arg-DHFRts is a heat-inducible degradation signal that can be used to produce ts mutants without a search for ts mutations.

In vivo half-life of a protein is a function of its amino-terminal residue.

When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.

The N-end rule in bacteria.

The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Distinct versions of the N-end rule operate in all eukaryotes examined. It is shown that the bacterium Escherichia coli also has the N-end rule pathway. Amino-terminal arginine, lysine, leucine, phenylalanine, tyrosine, and tryptophan confer 2-minute half-lives on a test protein; the other amino-terminal residues confer greater than 10-hour half-lives on the same protein. Amino-terminal arginine and lysine are secondary destabilizing residues in E. coli because their activity depends on their conjugation to the primary destabilizing residues leucine or phenylalanine by leucine, phenylalanine-transfer RNA-protein transferase. The adenosine triphosphate-dependent protease Clp (Ti) is required for the degradation of N-end rule substrates in E. coli.

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.

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.

The ubiquitin system.

Eukaryotes contain a highly conserved multi-enzyme system that covalently links ubiquitin to a variety of intracellular proteins that bear degradation signals recognized by this system. The resulting ubiquitin-protein conjugates are degraded by the 26S proteasome, a large ATP-dependent protease. Pathways that involve ubiquitin underlie a multitude of processes, including cell differentiation, the cell cycle and responses to stress.

A nuclease-hypersensitive region forms de novo after chromosome replication.

Regular nucleosome arrays in eucaryotic chromosomes are punctuated at specific locations, such as active promoters and replication origins, by apparently nucleosome-free sites, also called nuclease-hypersensitive, or exposed, regions. The -400-base pair-exposed region within simian virus 40 (SV40) chromosomes is present in approximately 20% of the chromosomes in lytically infected cells and encompasses the replication origin, transcriptional enhancer, and both late and early SV40 promoters. We report that nearly all SV40 chromosomes lacked the exposed region during replication and that newly formed chromosomes acquired the exposed region of the same degree as did bulk SV40 chromosomes within 1 h after replication. Furthermore, a much lower but significant level of exposure was detectable in late SV40 replication intermediates, indicating that formation of the exposed region could start within minutes after passage of the replication fork.

Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue. We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2. The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouse Ate1 gene. Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster) Ate1 genes encode a single form of ATE1p. A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p-GFP is exclusively cytosolic. Mouse ATE1-1p and ATE1-2p were examined by expressing them in ate1Delta Saccharomyces cerevisiae in the presence of test substrates that included Asp-betagal (beta-galactosidase) and Cys-betagal. Both forms of the mouse R-transferase conferred instability on Asp-betagal (but not on Cys-betagal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p. The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is approximately 0.1 in the skeletal muscle, approximately 0.25 in the spleen, approximately 3.3 in the liver and brain, and approximately 10 in the testis, suggesting that the two R-transferases are functionally distinct.

Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro.

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

Construction and analysis of mouse strains lacking the ubiquitin ligase UBR1 (E3alpha) of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3alpha). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Delta S. cerevisiae. We also constructed UBR1(-/-) mouse strains that lacked the UBR1 protein. UBR1(-/-) mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1(-/-) mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1(-/-) mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein, UBR1(-/-) fibroblasts contained the N-end rule pathway. Thus, UBR1(-/-) mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3alpha). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.

Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.

Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum.

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-alpha-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-alpha-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

Nucleosome arrangement in green monkey alpha-satellite chromatin. Superimposition of non-random and apparently random patterns.

We have studied the structure of tandemly repetitive alpha-satellite chromatin (alpha-chromatin) in African green monkey cells (CV-1 line), using restriction endonucleases and staphylococcal nuclease as probes. While more than 80% of the 172-base-pair (bp) alpha-DNA repeats have a HindIII site, less than 15% of the alpha-DNA repeats have an EcoRI site, and most of the latter alpha-repeats are highly clustered within the CV-1 genome. EcoRI and HindIII solubilize approximately 8% and 2% of the alpha-chromatin, respectively, under the conditions used. EcoRI is thus approximately 30 times more effective than HindIII in solubilizing alpha-chromatin, with relation to the respective cutting frequencies of HindIII and EcoRI on alpha-DNA. EcoRI and HindIII solubilize largely non-overlapping subsets of alpha-chromatin. The DNA size distributions of both EcoRI- and HindIII-solubilized alpha-chromatin particles peak at alpha-monomers. These DNA size distributions are established early in digestion and remain strikingly constant throughout the digestion with either EcoRI or HindIII. Approximately one in every four of both EcoRI- and HindIII-solubilized alpha-chromatin particles is an alpha-monomer. Two-dimensional (deoxyribonucleoprotein leads to DNA) electrophoretic analysis of the EcoRI-solubilized, sucrose gradient-fractionated alpha-oligonucleosomes shows that they do not contain "hidden" EcoRI cuts. Moreover, although the EcoRI-solubilized alpha-oligonucleosomes contain one EcoRI site in every 172-bp alpha-DNA repeat, they are completely resistant to redigestion with EcoRI. This striking difference between the EcoRI-accessible EcoRI sites flanking an EcoRI-solubilized alpha-oligonucleosome and completely EcoRI-resistant internal EcoRI sites in the same alpha-oligonucleosome indicates either that the flanking EcoRI sites occur within a modified chromatin structure or that an altered nucleosome arrangement in the vicinity of a flanking EcoRI site is responsible for its location in the nuclease-sensitive internucleosomal (linker) region. Analogous redigestions of the EcoRI-solubilized alpha-oligonucleosomes with either HindIII, MboII or HaeIII (both before and after selective removal of histone H1 by an exchange onto tRNA) produce a self-consistent pattern of restriction site accessibilities. Taken together, these data strongly suggest a preferred nucleosome arrangement within the EcoRI-solubilized subset of alpha-oligonucleosomes, with the centers of most of the nucleosomal cores being approximately 20 bp and approximately 50 bp away from the nearest EcoRI and HindIII sites, respectively, within the 172-bp alpha-DNA repeat. However, as noted above, the clearly preferred pattern of nucleosome arrangement within the EcoRI-solubilized alpha-oligonucleosomes is invariably violated at the ends of every such alpha-oligonucleosomal particle, suggesting at least a partially statistical origin of this apparently non-random nucleosome arrangement.(ABSTRACT TRUNCATED AT 400 WORDS)