RESUMEN
Correction to: Chapter 26 in: Rubén Alcázar and Antonio F. Tiburcio (eds.), Polyamines: Methods and Protocols, Methods in Molecular Biology, vol. 1694, https://doi.org/10.1007/978-1-4939-7398-9_26.
RESUMEN
Polyamines are essential poly-cations with vital functions in all cellular systems. Their levels are controlled by intricate regulatory feedback mechanisms. Abnormally high levels of polyamines have been linked to cancer. A rate-limiting enzyme in the biosynthesis of polyamines in fungi and higher eukaryotes is ornithine-decarboxylase (ODC). Its levels are largely controlled posttranslationally via ubiquitin-independent degradation mediated by ODC antizyme (OAZ). The latter is a critical polyamine sensor in a feedback control mechanism that adjusts cellular polyamine levels. Here, we describe an approach employing quantitative western blot analyses that provides in vivo evidence for cotranslational polyamine-sensing by nascent OAZ in yeast. In addition, we describe an in vitro method to detect polyamine binding by antizyme.
Asunto(s)
Poliaminas/metabolismo , Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/genética , Secuencia de Bases , Western Blotting , Unión Proteica , Proteínas/química , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Ornithine decarboxylase (ODC), a ubiquitin-independent substrate of the proteasome, is a homodimeric protein with a rate-limiting function in polyamine biosynthesis. Polyamines regulate ODC levels by a feedback mechanism mediated by ODC antizyme (OAZ). Higher cellular polyamine levels trigger the synthesis of OAZ and also inhibit its ubiquitin-dependent proteasomal degradation. OAZ binds ODC monomers and targets them to the proteasome. Here, we report that polyamines, aside from their role in the control of OAZ synthesis and stability, directly enhance OAZ-mediated ODC degradation by the proteasome. Using a stable mutant of OAZ, we show that polyamines promote ODC degradation in Saccharomyces cerevisiae cells even when OAZ levels are not changed. Furthermore, polyamines stimulated the in vitro degradation of ODC by the proteasome in a reconstituted system using purified components. In these assays, spermine shows a greater effect than spermidine. By contrast, polyamines do not have any stimulatory effect on the degradation of ubiquitin-dependent substrates.
RESUMEN
20S proteasomes constitute the proteolytic core of large protease complexes found in all branches of life. Among these, the eukaryotic 26S proteasome ubiquitously poses as a vital final entity in regulated degradation of intracellular proteins. The composition of 20S core particles has been disclosed in detail, facilitated by groundbreaking studies on ancestral prokaryotic 20S proteasomes of low complexity and culminated in the crystal structure determination of the much more complex eukaryotic particles. This article first summarizes insights into the structural organization of the 20S core followed by characterization of its proteolytic activities, which are confined to the central cavity of the particle. In eukaryotes they reside in three different subunit types differing in their preference for cleavage sites in substrates as well as in their importance for the proteasome's cellular function. The second part reviews current knowledge on the biogenesis pathways of 20S core particles, which have to ensure not only the fixed subunit arrangement but also activation of proteolytic subunits in a late assembly state.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Cisteína Endopeptidasas/inmunología , Células Eucariotas/metabolismo , Complejos Multienzimáticos/inmunología , Nanotecnología , Complejo de la Endopetidasa Proteasomal , Transporte de ProteínasRESUMEN
The assembly of individual mammalian proteasome subunits into catalytically active 20S proteasome is not well understood. Herein, we report the identification and characterization of human and mouse homologues of the yeast proteasome maturating factor Ump1p. We delineate the region of hUMP1 implicated in the specific interaction with proteasome precursors and show that hUMP1 protein is absent from the mature form of the 20S proteasome. We also show that the transcript level of mammalian UMP1 is increased after IFN-gamma treatment and that mammalian UMP1 is functionally related to but not interchangeable with its yeast homologue.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Chaperonas Moleculares/metabolismo , Complejos Multienzimáticos/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Clonación Molecular , Humanos , Ratones , Ratones Endogámicos C57BL , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal , Unión Proteica , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
Ligation of the ubiquitin-like protein SUMO (Smt3p) to other proteins is essential for viability of the yeast Saccharomyces cerevisiae. Like ubiquitin (Ub), SUMO undergoes ATP-dependent activation by a specific activating enzyme. SUMO-activating enzyme is a heterodimer composed of Uba2p and Aos1p, polypeptides with sequence similarities, respectively, to the C- and N-terminal parts of Ub-activating enzyme. To study the function of SUMO conjugation, we isolated uba2 mutants that were temperature-sensitive for growth. In these mutants conjugation of SUMO to other proteins was drastically reduced, even at the temperature permissive for growth. In a screen for spontaneous suppressors of the temperature-sensitive growth phenotype of the mutant uha2-ts9, we isolated a strain with a null mutation (sut9) in a gene of hitherto unknown function (SUT9/YIL031W/SMT4). This gene encodes a protein with similarities to Ulp1p, a dual-function protease that processes the SUMO precursor and deconjugates SUMO from its substrates. The novel protein was therefore termed Ulp2p. Inactivation of ULP2 in a strain expressing wild-type SUMO-activating enzyme resulted in slow and temperature-sensitive growth, and accumulation of SUMO conjugates. Thus, mutations in SUMO-activating enzyme and mutations in Ulp2p suppress each other, indicating that SUMO conjugation and deconjugation must be in balance for cells to grow normally. Other phenotypes of ulp2 mutants include a defect in cell cycle progression, hypersensitivity to DNA damage, and chromosome mis-segregation. Ulp2p is predominantly located within the nucleus, whereas Ulp1p colocalizes with nuclear pore complex proteins, indicating that the apparently distinct functions of the two SUMO deconjugating enzymes are spatially separated.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Enzimas Activadoras de Ubiquitina , Ubiquitinas/química , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , División Celular , Núcleo Celular/metabolismo , Daño del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas Fúngicas/genética , Genes Fúngicos , Humanos , Ligasas/genética , Ligasas/metabolismo , Datos de Secuencia Molecular , Mutación , Membrana Nuclear/metabolismo , Fenotipo , Proteína SUMO-1 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Supresión Genética , Temperatura , Ubiquitina-Proteína Ligasas , Ubiquitinas/genéticaRESUMEN
We report the discovery of a short-lived chaperone that is required for the correct maturation of the eukaryotic 20S proteasome and is destroyed at a specific stage of the assembly process. The S. cerevisiae Ump1p protein is a component of proteasome precursor complexes containing unprocessed beta subunits but is not detected in the mature 20S proteasome. Upon the association of two precursor complexes, Ump1p is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. Cells lacking Ump1p exhibit a lack of coordination between the processing of beta subunits and proteasome assembly, resulting in functionally impaired proteasomes. We also show that the propeptide of the Pre2p/Doa3p beta subunit is required for Ump1p's function in proteasome maturation.
Asunto(s)
Cisteína Endopeptidasas , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Ubiquitinas/metabolismo , Endopeptidasas/genética , Proteínas Fúngicas/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Mutagénesis , Complejo de la Endopetidasa Proteasomal , Precursores de Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Esporas Fúngicas/fisiología , Especificidad por SustratoRESUMEN
SMT3 is an essential Saccharomyces cerevisiae gene encoding a 11.5 kDa protein similar to the mammalian ubiquitin-like protein SUMO-1. We have found that Smt3p, like SUMO-1 and ubiquitin, can be attached to other proteins post-translationally and have characterized the processes leading to the activation of the Smt3p C-terminus for conjugation. First, the SMT3 translation product is cleaved endoproteolytically to expose Gly98, the mature C-terminus. The presence of Gly98 is critical for Smt3p's abilities to be conjugated to protein substrates and to complement the lethality of a smt3Delta strain. Smt3p undergoes ATP-dependent activation by a novel heterodimeric enzyme consisting of Uba2p, a previously identified 71 kDa protein similar to the C-terminus of ubiquitin-activating enzymes (E1s), and Aos1p (activation of Smt3p), a 40 kDa protein similar to the N-terminus of E1s. Experiments with conditional uba2 mutants showed that Uba2p is required for Smt3p conjugation in vivo. Furthermore, UBA2 and AOS1 are both essential genes, providing additional evidence that they act in a distinct pathway whose role in cell viability is to conjugate Smt3p to other proteins.
Asunto(s)
Ligasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Alelos , Secuencia de Aminoácidos , Sitios de Unión , Supervivencia Celular , Dimerización , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Glicina , Humanos , Datos de Secuencia Molecular , Unión Proteica , Procesamiento Proteico-Postraduccional , Receptores de Estrógenos/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina , Enzimas Activadoras de Ubiquitina , Ubiquitina-Proteína Ligasas , Ubiquitinas/químicaRESUMEN
A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Ligasas , Proteínas de Saccharomyces cerevisiae , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfatasas , Alelos , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación Puntual , Desnaturalización Proteica , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae , Relación Estructura-Actividad , Proteína que Contiene ValosinaRESUMEN
Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The "activated" Ub, linked via a thioester bond to a specific cysteine residue in one of several Ub-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins. In the yeast Saccharomyces cerevisiae, a 114-kDa E1 enzyme is encoded by an essential gene termed UBA1 (McGrath, J.P., Jentsch, S., and Varshavsky, A. (1991) EMBO J. 10, 227-236). We describe the isolation and analysis of another essential gene, termed UBA2, that encodes a 71-kDa protein with extensive sequence similarities to both the UBA1-encoded yeast E1 and E1 enzymes of other organisms. The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserved between the known E1 enzymes and contains the active-site cysteine of E1. This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involved a transient thioester bond between Uba2p and either Ub or another protein. Uba2p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Uba1p (E1 enzyme) and Uba2p cannot complement each others essential functions even if their subcellular localization is altered by mutagenesis. Uba2p appears to interact with itself and several other S. cerevisiae proteins with apparent molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitinated in vivo, suggesting that at least a fraction of Uba2p is metabolically unstable. Uba2p is likely to be a component of the Ub system that functions as either an E2 or E1/E2 enzyme.
Asunto(s)
Genes Fúngicos , Ligasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN de Hongos , Humanos , Ligasas/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Enzimas Activadoras de Ubiquitina , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Fúngicas/genética , Mutagénesis , Proteínas Recombinantes de Fusión/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/metabolismo , Semivida , Calor , Fenotipo , Saccharomyces cerevisiae/genética , Temperatura , Tetrahidrofolato Deshidrogenasa/metabolismo , UbiquitinasRESUMEN
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, substrates of the N-end rule pathway are targeted for degradation by a complex that includes the 225-kDa N-recognin, encoded by UBR1, and the 20-kDa ubiquitin-conjugating enzyme encoded by UBC2. We report that both physical stability and functional activity of the N-recognin.Ubc2 complex require the presence of a highly acidic 23-residue region at the C terminus of Ubc2. Ubc2-C88A, an inactive variant of Ubc2 in which the active-site Cys-88 has been replaced by Ala, is shown to retain the affinity for N-recognin. Expression of Ubc2-C88A inhibits the N-end rule pathway, apparently as a result of competition between Ubc2 and Ubc2-C88A for binding to N-recognin. The two-hybrid (interaction cloning) technique was used to identify a approximately 170-residue C-terminal fragment of the 1,950-residue N-recognin as a Ubc2-interacting domain. We also show that the level of UBR1 mRNA decreases upon overexpression of UBC2. This effect of UBC2 is observed with cells whose UBR1 is expressed from an unrelated promoter but is not observed if UBR1 contains a frameshift mutation, or if the Ubc2 protein lacks its C-terminal acidic region. The N-recognin.Ubc2 complex appears to regulate the expression of N-recognin through changes in the metabolic stability of its mRNA.
Asunto(s)
Proteínas Fúngicas/metabolismo , Ligasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas , Alelos , Northern Blotting , Clonación Molecular , Proteínas Fúngicas/genética , Marcadores Genéticos , Ligasas/genética , Plásmidos , Unión Proteica , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas Recombinantes/metabolismo , Enzimas Ubiquitina-ConjugadorasRESUMEN
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 organisms examined, from mammals to bacteria. We show that UBC2(RAD6), one of at least seven ubiquitin-conjugating enzymes in the yeast Saccharomyces cerevisiae, is essential for multiubiquitination and degradation of the N-end rule substrates. We also show that UBC2 is physically associated with UBR1, the recognition component of the N-end rule pathway. These results indicate that some of the UBC2 functions, which include DNA repair, induced mutagenesis, sporulation, and regulation of retrotransposition, are mediated by protein degradation via the N-end rule pathway.
Asunto(s)
Ligasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Reparación del ADN , Proteínas Fúngicas/metabolismo , Haploidia , Ligasas/genética , Modelos Biológicos , Unión Proteica , Saccharomyces cerevisiae/genética , Enzimas Ubiquitina-Conjugadoras , Ubiquitinas/metabolismoRESUMEN
The Schwanniomyces occidentalis glucoamylase (GAM)-encoding gene (GAM1) was isolated from a lambda Charon4A genomic library using synthetic oligodeoxynucleotides as probes. GAM1 contains an ORF of 2874 nucleotides (nt) coding for 958 amino acids. S1 mapping revealed that the transcript has only a very short 5'-untranslated leader of 8-12 nt. Disruption and displacement of the GAM1 gene in Sc. occidentalis resulted in loss of the ability to grow on starch efficiently. The gam1 strains still exhibit low GAM activity suggesting that at least a second weakly expressed GAM-encoding gene (GAM2) is present in Sc. occidentalis. Expression of the Sc. occidentalis GAM1 gene in Saccharomyces cerevisiae was achieved after promoter exchange. S. cerevisiae cells transformed with centromere plasmids carrying the GAM1 gene fused to promoters of different S. cerevisiae genes, namely GAL1, PDC1 and ADH1, efficiently secrete GAM and are able to grow with soluble starch as a sole carbon source. The essential enzymatic properties of the GAMs secreted from S. cerevisiae and Sc. occidentalis are identical, although the modifications of the proteins are different.
Asunto(s)
Genes Fúngicos , Glucano 1,4-alfa-Glucosidasa/genética , Levaduras/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , Expresión Génica , Glucano 1,4-alfa-Glucosidasa/metabolismo , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Transformación GenéticaRESUMEN
We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.
Asunto(s)
Ascomicetos/enzimología , Transformación Genética , Levaduras/enzimología , alfa-Amilasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Expresión Génica , Datos de Secuencia Molecular , alfa-Amilasas/análisis , alfa-Amilasas/metabolismoRESUMEN
High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called "SwARS1". The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5-10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned alpha-amylase gene from S. occidentalis secreted about five times more alpha-amylase than cells without additional copies of the alpha-amylase gene. Both the chromosomal copy and the plasmid-carried copies of the alpha-amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.