RESUMO
Hub1/Ubl5 is a member of the family of ubiquitin-like proteins (UBLs). The tertiary structure of Hub1 is similar to that of ubiquitin; however, it differs from known modifiers in that there is no conserved glycine residue near the C terminus which, in ubiquitin and UBLs, is required for covalent modification of target proteins. Instead, there is a conserved dityrosine motif proximal to the terminal nonconserved amino acid. In S. cerevisiae, high molecular weight adducts can be formed in vivo from Hub1, but the structure of these adducts is not known, and they could be either covalent or noncovalent. The budding yeast HUB1 gene is not essential, but Delta hub1 mutants display defects in mating. Here, we report that fission yeast hub1 is an essential gene, whose loss results in cell cycle defects and inefficient pre-mRNA splicing. A screen for Hub1 interactors identified Snu66, a component of the U4/U6.U5 tri-snRNP splicing complex. Furthermore, overexpression of Snu66 suppresses the lethality of a hub1ts mutant. In cells lacking functional hub1, the nuclear localization of Snu66 is disrupted, suggesting that an important role for Hub1 is the correct subcellular targeting of Snu66, although our data suggest that Hub1 is likely to perform other roles in splicing as well.
Assuntos
Splicing de RNA/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclo Celular/fisiologia , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Genes Essenciais/fisiologia , Microscopia de Fluorescência , Mutação/genética , Oligonucleotídeos , Transporte Proteico/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Técnicas do Sistema de Duplo-Híbrido , Complexos Ubiquitina-Proteína Ligase/genética , UbiquitinasRESUMO
The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain. This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes. Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome. A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain. A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome. We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding. We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes.
Assuntos
Proteínas de Ciclo Celular , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Ligação Competitiva , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ligantes , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Type I ubiquitin-like proteins constitute a family of protein modifiers. Here we report the identification of a posttranslational protein modifier from Saccharomyces cerevisiae, Hub1. Overexpression of Hub1 resulted in enhanced conjugate formation when its carboxyl-terminal residue was deleted, suggesting that mature Hub1 may be produced by proteolytic processing. In vivo targets of Hub1 conjugation included cell polarity factors Sph1 and Hbt1. In the hub1Delta mutant, the subcellular localization of both Hbt1 and Sph1 was disrupted, and cell polarization during the formation of mating projections was defective. Consistent with these polarization defects, the hub1Delta mutant was deficient in mating.
