Ubiquitin chains serve as a recognition motif for the proteasome, a multisubunit protease, which degrades its substrates into polypeptides while releasing ubiquitin for reuse. Yeast proteasomes contain two deubiquitinating enzymes, Ubp6 and Rpn11. Rpn11 promotes protein breakdown through its degradation-coupled activity. In contrast, we show here that Ubp6 has the capacity to delay the degradation of ubiquitinated proteins by the proteasome. However, delay of degradation by Ubp6 does not require its catalytic activity, indicating that Ubp6 has both deubiquitinating activity and proteasome-inhibitory activity. Delay of degradation by Ubp6 appears to provide a time window allowing gradual deubiquitination of the substrate by Ubp6. Rpn11 catalyzes en bloc chain removal, and Ubp6 interferes with degradation at or upstream of this step, so that degradation delay by Ubp6 is accompanied by a switch in the mode of ubiquitin chain processing. We propose that Ubp6 regulates both the nature and magnitude of proteasome activity.

The ubiquitin ligase Hul5 was recently identified as a component of the proteasome, a multisubunit protease that degrades ubiquitin-protein conjugates. We report here a proteasome-dependent conjugating activity of Hul5 that endows proteasomes with the capacity to extend ubiquitin chains. hul5 mutants show reduced degradation of multiple proteasome substrates in vivo, suggesting that the polyubiquitin signal that targets substrates to the proteasome can be productively amplified at the proteasome. However, the products of Hul5 conjugation are subject to disassembly by a proteasome-bound deubiquitinating enzyme, Ubp6. A hul5 null mutation suppresses a ubp6 null mutation, suggesting that a balance of chain-extending and chain-trimming activities is required for proper proteasome function. As the association of Hul5 with proteasomes was found to be strongly stabilized by Ubp6, these enzymes may be situated in proximity to one another. We propose that through dynamic remodeling of ubiquitin chains, proteasomes actively regulate substrate commitment to degradation.

Nob1p is a nuclear protein that forms a complex with the 19S regulatory particle of the 26S proteasome and with uncharacterized nuclear protein Pno1p. Overexpression of NOB1 overrode the defects in maturation of the 20S proteasome of ump1⌬ cells, and temperature-sensitive nob1 and pno1 mutants exhibited defects in the processing of the ␤ subunits and in the assembly of the 20S and the 26S proteasomes. A defect in either NOB1 or PNO1 caused accumulation of newly formed Pre6p in the cytoplasm, whereas Pre6p of the ump1⌬ strain accumulated in the nucleus irrespective of the temperature. Here we present a model proposing that (1) Nob1p serves as a chaperone to join the 20S proteasome with the 19S regulatory particle in the nucleus and facilitates the maturation of the 20S proteasome and degradation of Ump1p, and (2) Nob1p is then internalized into the 26S proteasome and degraded to complete 26S proteasome biogenesis.

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Overexpression of the mammalian proapoptotic protein Bax induces cell death in plant and yeast cells. The Bax inihibitor-1 (BI-1) gene rescues yeast and plant from Bax-mediated lethality. Using the Arabidopsis BI-1 (AtBI-1) gene controlled by the GAL1 promoter as a cell death suppressor in yeast, Cdf1 (cell growth defect factor-1) was isolated from Arabidopsis cDNA library. Overexpression of Cdf1 caused cell death in yeast, whereas such an effect was suppressed by co-expression of AtBI-1. The Cdf1 protein fused with a green fluorescent protein was localized in the mitochondria and resulted in the loss of mitochondrial membrane potential in yeast. The Bax-resistant mutant BRM1 demonstrated tolerance against Cdf1-mediated lethality, whereas the ⌬atp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.Proteins encoded by Bcl-2 family interact with each other and either promote or inhibit metazoan apoptosis. A basic local alignment search tool (BLAST) data base search of the Arabidopsis thaliana genome showed no obvious homologues of any crucial regulator of metazoan apoptosis (members of the Bcl-2 family, p53, etc.) (1, 2). However, it has been demonstrated that mammalian proapoptotic proteins can kill plant (3, 4) as well as yeast (5-9). Furthermore, the antiapoptotic proteins Bcl-XL, Bcl-2, and Ced9 protect tobacco plants against cell death induced by ultraviolet light irradiation, paraquat treatment, hypersensitive response upon tobacco mosaic virus infection, and fungal pathogens (10, 11). The Pseudomonas AvrPtoB effecter protein, conserved among diverse genera of plant pathogens, acts to inhibit Bax-induced cell death (12). This evidence clearly suggests that some mechanisms of cell death are evolutionarily conserved throughout the metazoa and plants.In yeast, cell death with apoptosis-like features has also been reported after treatment with acetic acid, UV light irradiation, and H 2 O 2 (reviewed in Ref. 13). Madeo et al. (14) reported that a caspase-like gene (Yor197w) identified in Saccharomyces cerevisiae was implicated in cell death induced by H 2 O 2 , acetic acid, and aging. Yeast cells undergoing Bax-induced death exhibit ultrastructural changes that include massive cytosolic vacuolation and apparent disruption of mitochondria (6,8).Classical yeast genetic approaches have been successfully applied for identification of genes related to the regulation of mammalian apoptosis. Even though the yeast genome lacks some of the molecular machinery responsible for apoptosis in metazoans, it can be powerful tool in the isolation of apoptosis-related genes.The Bax inhibitor-1 (BI-1) 2 gene of Arabidopsis (AtBI-1) and the Oryza sativa Bax inhibitor-1 gene (OsBI-1), which are plant homologues of mammalian antiapoptotic gene BI-1, were isolated as suppressors of Bax-mediated lethality in yeast (15)(16)(17)(18)(19). Mammalian BI-1 suppresses apoptosis induced by Bax, etoposide, staurosporine, and growth factor depriva...