RESUMEN
A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P. This also depends on the ribosome-associated E3 ligase Ltn1 and its co-factor Rqc1. Although Hsp104 is absolutely required for proteasomal degradation of Sdj1-L169P aggregates, the degradation of already aggregated Sdj1-L169P occurs independently of Ltn1 and Rqc1. Thus, our data point to soluble Sdj1-L169P being targeted early by Ltn1 and Rqc1. The fraction of Sdj1-L169P that escapes this first inspection then forms aggregates that are subsequently cleared via an Hsp104- and proteasome-dependent pathway.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Coenzimas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Oncogénicas/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Coenzimas/química , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas , Proteína Desglicasa DJ-1 , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteolisis , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26S proteasome. The proteasome subunits Rpn10 and Rpn13 are known to bind ubiquitin, but genetic and biochemical data suggest the existence of at least one other substrate receptor. Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and K48. Atomic resolution data show that Dss1 is disordered and binds ubiquitin by binding sites characterized by acidic and hydrophobic residues. The complementary binding region in ubiquitin is composed of a hydrophobic patch formed by I13, I44, and L69 flanked by two basic regions. Mutations in the ubiquitin-binding site of Dss1 cause growth defects and accumulation of ubiquitylated proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Ubiquitina/metabolismo , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión al ARN , Proteínas de Schizosaccharomyces pombe/química , Ubiquitina/químicaRESUMEN
BACKGROUND: In mammalian cells, ASPL is involved in insulin-stimulated redistribution of the glucose transporter GLUT4 and assembly of the Golgi apparatus. Its putative yeast orthologue, Ubx4, is important for proteasome localization, endoplasmic reticulum-associated protein degradation (ERAD), and UV-induced degradation of RNA polymerase. RESULTS: Here, we show that ASPL is a cofactor of the hexameric ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast. In addition, ASPL interacts in vitro with NSF, another hexameric ATPase complex. ASPL localizes to the ER membrane. The central area in ASPL, containing both a SHP box and a UBX domain, is required for binding to the p97 N-domain. Knock-down of ASPL does not impair degradation of misfolded secretory proteins via the ERAD pathway. Deletion of UBX4 in yeast causes cycloheximide sensitivity, while ubx4 cdc48-3 double mutations cause proteasome mislocalization. ASPL alleviates these defects, but not the impaired ERAD. CONCLUSIONS: In conclusion, ASPL and Ubx4 are homologous proteins with only partially overlapping functions. Both interact with p97/Cdc48, but while Ubx4 is important for ERAD, ASPL appears not to share this function.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/análisis , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Eliminación de Gen , Técnicas de Inactivación de Genes , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mutación , Proteínas Nucleares/análisis , Proteínas de Fusión Oncogénica/análisis , Proteínas de Fusión Oncogénica/genética , Complejo de la Endopetidasa Proteasomal/análisis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Conjugation of the ubiquitin-like modifier Nedd8 to cullins is critical for the function of SCF-type ubiquitin ligases and thus facilitates ubiquitin conjugation and ultimately degradation of SCF substrates, including several cell cycle regulators. Like ubiquitin, Nedd8 is produced as a precursor that must first be processed before it becomes active. In Saccharomyces cerevisiae this is carried out exclusively by the enzyme Yuh1. RESULTS: Here we show that in the fission yeast, Schizosaccharomyces pombe, the Yuh1 orthologue, Uch1, is not the sole Nedd8 processing enzyme. Instead it appears that deubiquitylating enzymes can efficiently process the Nedd8 precursor in vivo. CONCLUSIONS: Several enzymes contribute to Nedd8 precursor processing including a number of deubiquitylating enzymes.
