RESUMEN
The stalk proteins P1 and P2, which are fundamental for ribosome activity, are the only ribosomal components for which there is a cytoplasmic pool. Accumulation of these two proteins is differentially regulated in Saccharomyces cerevisiae by degradation. In the absence of P2, the amount of P1 is drastically reduced; in contrast, P2 proteins are not affected by a deficiency in P1. However, association with P2 protects P1 proteins. The half-life of P1 is a few minutes, while that of P2 is several hours. The proteasome is not involved in the degradation of P1 proteins. The different sensitivity to degradation of these two proteins is associated with two structural features: phosphorylation and N-terminus structure. A phosphorylation site at the C-terminus is required for P1 proteolysis. P2 proteins, despite being phosphorylated, are protected by their N-terminal peptide. An exchange of the first five amino acids between the two types of protein makes P1 resistant and P2 sensitive to degradation.
Asunto(s)
Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Secuencia de Bases , Sitios de Unión , Quimera/genética , Cisteína Endopeptidasas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expresión Génica , Semivida , Peso Molecular , Complejos Multienzimáticos/metabolismo , Oligodesoxirribonucleótidos/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/genética , Empalme del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas , Saccharomyces cerevisiae/genéticaRESUMEN
The yeast ribosomal stalk is formed by a protein pentamer made of the 38 kDa P0 and four 12 kDa acidic P1/P2. The interaction of recombinant acidic proteins P1 alpha and P2 beta with ribosomes from Saccharomyces cerevisiae D4567, lacking all the 12 kDa stalk components, has been used to study the in vitro assembly of this important ribosomal structure. Stimulation of the ribosome activity was obtained by incubating simultaneously the particles with both proteins, which were nonphosphorylated initially and remained unmodified afterward. The N-terminus state, free or blocked, did not affect either the binding or reactivating activity of both proteins. Independent incubation with each protein did not affect the activity of the particles, however, protein P2 beta alone was unable to bind the ribosome whereas P1 alpha could. The binding of P1 alpha alone is a saturable process in acidic-protein-deficient ribosomes and does not take place in complete wild-type particles. Binding of P1 proteins in the absence of P2 proteins takes also place in vivo, when protein P1 beta is overexpressed in S. cerevisiae. In contrast, protein P2 beta is not detected in the ribosome in the P1-deficient D67 strain despite being accumulated in the cytoplasm. The results confirm that neither phosphorylation nor N-terminal blocking of the 12 kDa acidic proteins is required for the assembly and function of the yeast stalk. More importantly, and regardless of the involvement of other elements, they indicate that stalk assembling is a coordinated process, in which P1 proteins would provide a ribosomal anchorage to P2 proteins, and P2 components would confer functionality to the complex.
Asunto(s)
Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfoproteínas/genética , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/fisiología , Saccharomyces cerevisiae/genéticaRESUMEN
Mutants of the yeast Saccharomyces cerevisiae defective in the RAD17 gene are sensitive to ultraviolet (UV) and gamma radiation and manifest a defect in G2 arrest following radiation treatment. We have cloned the RAD17 gene by complementation of the UV sensitivity of a rad17-1 mutant and identified an ORF of 1.2 kb encoding a predicted gene product of 45.4 kDa with homology to the Schizosaccharomyces pombe rad1+ gene product and to Ustilago maydis Rec1, a known 3'->5'exonuclease. The RAD17 transcript is cell cycle regulated, with maximum steady-state levels during late G1. The rad17-1 mutation represents a missense mutation that maps to a conserved region of the gene. A rad17 disruption mutant grows normally and manifests levels of UV sensitivity similar that of the rad17-1 strain. As previously observed with other genes involved in G2 arrest (such as RAD9 and RAD24), RAD17 regulates radiation-induced G1 checkpoints at at least two possible arrest stages. One is equivalent to or upstream of START, the other at or downstream of the Cdc4 execution point. However, the temperature sensitivity of the cell cycle mutant dna1-1 (a G1 arrest mutant) is not influenced by inactivation of RAD17.