RESUMO
Short-lived proteins are targeted for turnover by sequence elements known as degradation signals. Because of the large size and heterogeneity of these signals, the structural features important for their function are not well defined. In this study, we have isolated three classes of degradation signals by screening short artificial sequences for the ability to destabilize a reporter protein. Class I and class II signals were derived by inserting random nonapeptide sequences after the second residue of beta-galactosidase. Class III signals contained five-residue homopolymers at the same position. Class I beta-galactosidase turnover was inhibited in mutants lacking either the ubiquitin-conjugating enzyme Ubc2 or the ubiquitin protein ligase Ubr1. Class I random inserts functioned to promote N-terminal proteolytic processing and define a novel pathway for exposure of residues that are destabilizing according to the N-end rule. Efficient degradation of proteins containing class II signals required at least three Ubc enzymes: Ubc6, Ubc7, and either one of the related enzymes Ubc4 and Ubc5. Analysis of 56 amino acid substitutions in the class II signal suggested that it is recognized in the form of an amphipathic alpha helix. Class III signals consisted of short tracts of hydrophobic residues such as Leu and Ile. Degradation of class III proteins involved the Ubc4 and Ubc5 enzymes but not Ubc2, Ubc6, or Ubc7. Clusters of hydrophobic residues appear to be critical for the recognition of both class II and class III signals.
Assuntos
Ligases/metabolismo , Oligopeptídeos/metabolismo , Ubiquitinas/metabolismo , beta-Galactosidase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Biblioteca Gênica , Genes Reporter , Hidrólise , Ligases/classificação , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/classificação , Oligopeptídeos/genética , Engenharia de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , beta-Galactosidase/genéticaRESUMO
The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin. To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg. Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates. The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitin-conjugating enzyme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair. However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product. The UbK63R mutation may therefore define a new role of ubiquitin in DNA repair. The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.
Assuntos
Reparo do DNA , Lisina , Mutação Puntual , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Immunoblotting , Cinética , Ligases/metabolismo , Metanossulfonato de Metila/farmacologia , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Enzimas de Conjugação de Ubiquitina , Ubiquitinas/biossíntese , Ubiquitinas/química , Raios UltravioletaRESUMO
The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.
Assuntos
Ciclo Celular , Proteínas Fúngicas/metabolismo , Ubiquitinas/metabolismo , Endopeptidases/metabolismo , Genes Dominantes , Teste de Complementação Genética , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiaeRESUMO
Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclinas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Ciclo Celular , Ciclinas/biossíntese , Ciclinas/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Genes Fúngicos , Cinética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fosforilação , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , beta-Galactosidase/biossíntese , beta-Galactosidase/metabolismoRESUMO
The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.
Assuntos
Proteínas de Arabidopsis , Proteínas de Transporte/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Arabidopsis/metabolismo , Sequência de Bases , Proteínas de Transporte/química , Clonagem Molecular , Primers do DNA , Drosophila , Temperatura Alta , Humanos , Cinética , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Raios UltravioletaRESUMO
Plasmids containing cDNA copies of mRNAs induced in HeLa cells by heat shock have been isolated and characterized. In vitro translation of RNAs selected by hybridization to plasmid DNAs identified sequences representing the three major classes (89, 70 and 27-kDa) of heat-shock proteins (hsp) and a 60-kDa minor hsp. Plasmids with inserts specific for the 27, 60, and 70-kDa hsp each hybridize with a single discrete size class of heat-inducible mRNA. Plasmids specific for the 89-kDa protein, however, hybridize with either a 2.7- or 2.95-kb mRNA species. Both mRNAs are coordinately induced during heat shock. We show that the characteristic pattern of induction and repression of each class of hsp during sustained hyperthermia is the result of changes in the steady state level of each mRNA.
Assuntos
Clonagem Molecular , DNA/metabolismo , Genes , Proteínas de Choque Térmico/genética , RNA Mensageiro/genética , Transcrição Gênica , Enzimas de Restrição do DNA , Células HeLa/metabolismo , Temperatura Alta , Humanos , Hibridização de Ácido Nucleico , Plasmídeos , Biossíntese de ProteínasRESUMO
Differential scanning calorimetry was used to monitor the thermal transitions of the 70 kDa heat shock cognate protein (Hsc70). Hsc70 had endothermic transitions with midpoints (Tm) at 59 degrees C and 63 degrees C in the absence and presence of ATP, respectively, and a similar increase in Tm was observed using intrinsic fluorescence of tryptophan. Combined with increased exposure at 60 degrees C of non-polar residues of Hsc70 to which the hydrophobic, fluorescent probe ANS bound, these data indicate that the endotherms represent thermal denaturation and that bound nucleotide stabilizes Hsc70. An exothermic transition (Tm = 66 degrees C) was detected by calorimetry for Hsc70-apocytochrome c (apo c) complexes. An increase in intrinsic fluorescence with the same Tm and increased turbidity indicated aggregation of the denatured Hsc70-apo c. A novel finding was an exothermic transition of Hsc70 beginning at about 30 degrees C (Tm = 41 degrees C). No changes in either intrinsic fluorescence or ANS fluorescence attributable to protein transitions were detected in this temperature range. Examination of samples run on native polyacrylamide gels indicated that this exothermic transition was not due to Hsc70 aggregation or multimer formation. However, Hsc70 was protease-resistant at 20 degrees C, sensitive at 40 degrees C and resistant when returned to 20 degrees C, indicating that this exotherm is associated with a reversible conformational change. As an assay for Hsc70 chaperoning function, complex formation was measured as a function of temperature using a variety of substrates including the model unfolded protein apo c, a pigeon cytochrome c fragment, a representative hydrophobic-aromatic peptide FYQLALT, and a representative hydrophobic-basic motif NIVRKKK. For all of these substrates, the amount of complex formed increased with increasing temperature over the same range as the 41 degrees C exotherm. It is proposed that a conformational change exposes polar and charged residues in Hsc70 which subsequently become hydrated, resulting in an active chaperone. Hsc70 may be a thermal sensor that matches the supply of chaperoning activity with demand for it over the physiological temperature range of mammalian cells. Thermal activation of Hsc70 may also have a role in acquired thermotolerance.
Assuntos
Temperatura Corporal/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP70/fisiologia , Resposta ao Choque Térmico/fisiologia , Sequência de Aminoácidos , Animais , Apolipoproteínas C/metabolismo , Varredura Diferencial de Calorimetria , Proteínas de Transporte/análise , Bovinos , Fluorometria , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/análise , Chaperonas Moleculares/análise , Estrutura Molecular , Conformação Proteica , Coloração pela Prata , TriptofanoRESUMO
The mammalian 70-kilodalton heat shock cognate protein (Hsc70) is an abundant, cytosolic molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis. In vitro, purified Hsc70 was found to have a slow, intrinsic ATPase activity in the absence of protein substrates. The addition of an unfolded protein such as apocytochrome c stimulated ATP hydrolysis 2-3-fold. In contrast, the native holoprotein, cytochrome c, did not stimulate the ATPase rate, in accord with recent observations that 70-kilodalton heat shock proteins interact selectively with unfolded proteins. Stimulation of ATP hydrolysis by apocytochrome c was due to an increase in the Vmax, with no effect on the Km for ATP. Following hydrolysis of [3H]ATP, a relatively stable [3H]ADP.Hsc70 complex was formed. Release of [3H]ADP from Hsc70 was most efficient in the presence of other nucleotides such as ADP or ATP, suggesting that ADP release occurs as an ADP/ATP exchange reaction. The loss of radiolabeled ADP from Hsc70 in the presence of exogenous nucleotides followed first-order kinetics. In the presence of nucleotides, apocytochrome c induced a 2-fold increase in the rate of ADP release from Hsc70. Moreover, rate constants of the nucleotide exchange reaction measured in the absence and presence of apocytochrome c (0.16 and 0.34 min-1, respectively) closely matched the kcat values derived from ATP hydrolysis measurements (0.15 and 0.38 min-1, respectively). The results suggest that ADP release in a rate-limiting step in the Hsc70 ATPase reaction and that unfolded proteins stimulate ATP hydrolysis by accelerating the rate of ADP/ATP exchange.
Assuntos
Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP70 , Proteínas/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Apoproteínas/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Chaperoninas , Grupo dos Citocromos c/metabolismo , Citocromos c , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Cinética , Cloreto de Potássio/farmacologia , Dobramento de Proteína , Proteínas/efeitos dos fármacosRESUMO
Incubation of HeLa cells at 42 degrees C results in pronounced inhibition of the accumulation of 18S and 28S ribosomal RNA (rRNA) and non-heat shock polyadenylated messenger RNA (mRNA) in the cytoplasm. Accumulation of transfer RNA and 5S ribosomal RNA is not affected. Transcription of rRNA precursor is reduced to approximately 50% of the 37 degrees C rate after 10 min of hyperthermia and declines to 30% of the control rate after 1 hr. In contrast, the accumulation of mature rRNA in the cytoplasm is inhibited more than 95%. Quantitative hybridization experiments and Northern blot analysis detect little accumulation of rRNA precursor sequences in nuclei, suggesting that the majority of the rRNA that is synthesized is degraded. Heat stress at 42 degrees C was found to have little effect on transcription of most non-heat shock mRNAs. However, accumulation of individual non-heat shock mRNAs in the cytoplasm proceeds at reduced rates. These results indicate that the primary effect of elevated temperature on RNA metabolism in mammalian cells is inhibition of processing and/or transport. Despite this, steady-state levels of abundant and rapidly turning over mRNA species remain unchanged during prolonged heat stress. We find that the half-life of c-myc mRNA increases greater than twofold at 42 degrees C. Thus, 42 degrees C heat stress appears to inhibit both accumulation and turnover of non-heat shock mRNA.
Assuntos
Células HeLa/metabolismo , Temperatura Alta , RNA/metabolismo , Citoplasma/análise , Humanos , Oncogenes , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 28S/metabolismo , Fatores de Tempo , Transcrição Gênica , Uridina/metabolismoRESUMO
Heat shock proteins are rapidly synthesized when cells are exposed to stressful agents that cause protein damage. The 70-kDa heat shock induced proteins and their closely related constitutively expressed cognate proteins bind to unfolded and aberrant polypeptides and to hydrophilic peptides. The structural features of the 70-kDa heat shock proteins that confer the ability to associate with diverse polypeptides are unknown. In this study, we have used circular dichroism (CD) spectroscopy and secondary structure prediction to analyze the secondary structure of the mammalian 70-kDa heat shock cognate protein (hsc 70). The far-ultraviolet CD spectrum of hsc 70 indicates a large fraction of alpha-helix in the protein and resembles the spectra one obtains from proteins of the alpha/beta structural class. Analysis of the CD spectra with deconvolution methods yielded estimates of secondary structure content. The results indicate about 40% alpha-helix and 20% aperiodic structure within hsc 70 and between 16-41% beta-sheet and 21-0% beta-turn. The Garnier-Osguthorpe-Robson method of secondary structure prediction was applied to the rat hsc 70 amino acid sequence. The predicted estimates of alpha-helix and aperiodic structure closely matched the values derived from the CD analysis, whereas the predicted estimates of beta-sheet and beta-turn were midway between the CD-derived values. Present evidence suggests that the polypeptide ligand binding domain of the 70-kDa heat shock protein resides within the C-terminal 160 amino acids [Milarski, K. L., & Morimoto, R. I. (1989) J. Cell Biol. 109, 1947-1962].(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Choque Térmico/química , Sequência de Aminoácidos , Animais , Química Encefálica , Bovinos , Dicroísmo Circular , Proteínas de Choque Térmico/isolamento & purificação , Dados de Sequência Molecular , Conformação Proteica , RatosRESUMO
The 26 S proteasome is a multisubunit proteolytic complex responsible for degrading eukaryotic proteins targeted by ubiquitin modification. Substrate recognition by the complex is presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through lysine 48. We have identified previously a candidate for one such receptor from diverse species, designated here as Mcb1 for Multiubiquitin chain-binding protein, based on its ability to bind Lys48-linked multiubiquitin chains and its location within the 26 S proteasome complex. Even though Mcb1 is likely not the only receptor in yeast, it is necessary for conferring resistance to amino acid analogs and for degrading a subset of ubiquitin pathway substrates such as ubiquitin-Pro-beta-galactosidase (Ub-Pro-beta-gal) (van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R. D. (1996) Mol. Cell. Biol. 16, 6020-28). To further define the role of Mcb1 in substrate recognition by the 26 S proteasome, a structure/function analysis of various deletion and site-directed mutants of yeast and Arabidopsis Mcb1 was performed. From these studies, we identified a single stretch of conserved hydrophobic amino acids (LAM/LALRL/V (ScMcb1 228-234 and At-Mcb1 226-232)) within the C-terminal half of each polypeptide that is necessary for interaction with Lys48-linked multiubiquitin chains. Unexpectedly, this domain was not essential for either Ub-Pro-beta-gal degradation or conferring resistance to amino acid analogs. The domain responsible for these two activities was mapped to a conserved region near the N terminus. Yeast and Arabidopsis Mcb1 derivatives containing an intact multiubiquitin-binding site but missing the N-terminal region failed to promote Ub-Pro-beta-gal degradation and even accentuated the sensitivity of the yeast delta mcb1 strain to amino acid analogs. This hypersensitivity was not caused by a gross defect in 26 S proteasome assembly as mutants missing either the N-terminal domain or the multiubiquitin chain-binding site could still associate with 26 S proteasome and generate a complex indistinguishable in size from that present in wild-type yeast. Together, these data indicate that residues near the N terminus, and not the multiubiquitin chain-binding site, are most critical for Mcb1 function in vivo.
Assuntos
Proteínas de Arabidopsis , Proteínas de Transporte/química , Peptídeo Hidrolases/química , Complexo de Endopeptidases do Proteassoma , Sequência de Aminoácidos , Animais , Arabidopsis , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Sequência Consenso , Análise Mutacional de DNA , Primers do DNA , Drosophila , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , LevedurasRESUMO
The 26S proteasome is a 2-Megadalton proteolytic complex with over 30 distinct subunits. The 19S particle, a subcomplex of the 26S proteasome, is thought to confer ATP-dependence and ubiquitin-dependence on the proteolytic core particle of the proteasome. Given the complexity of the 19S particle, genetic approaches are likely to play an important role in its analysis. We have initiated biochemical and genetic studies of the 19S particle in Saccharomyces cerevisiae. Here we describe the localization to the proteasome of several ATPases that were previously proposed to be involved in transcription. Independent studies indicate that the mammalian 26S proteasome contains closely related ATPases. We have also found that the multiubiquitin chain binding protein Mcb1, a homolog of the mammalian S5a protein, is a subunit of the yeast proteasome. However, contrary to expectation, MCB1 is not an essential gene in yeast. The mcb1 mutant grows at a nearly wild-type rate, and the breakdown of most ubiquitin-protein conjugates is unaffected in this strain. One substrate, Ub-Proline-beta gal, was found to require MCB1 for its breakdown, but it remains unclear whether Mcb1 serves as a ubiquitin receptor in this process. Our data suggest that the recognition of ubiquitin conjugates by the proteasome is a complex process which must involve proteins other than Mcb1.