Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities.

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.

Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation.

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.

A novel Arabidopsis gene causes Bax-like lethality in Saccharomyces cerevisiae.

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 Deltaatp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.

The inhibitory effect of luteolin-7-O-glucoside on the formation of pentyl and 7-carboxyheptyl radicals from 13-hydroperoxy-9,11-octadecadienoic acid in the presence of iron(II) ions.

A flavone glucoside, luteolin-7-O-glucoside (luteolin-7-G) inhibited the formation of pentyl and 7-carboxyheptyl radicals in the reaction of 13-hydroperoxy-9,11-octadecadienoic (13-HPODE) acid with iron(II) ions. The inhibitory effect of luteolin-7-G was diminished in the presence of EDTA. These results indicated that the inhibitory effects of luteolin-7-G occur partly through the chelation of iron ions. Measurement of visible spectra also showed that luteolin-7-G chelates iron ions. On the other hand, luteolin-7-G did not inhibit the reaction under anaerobic conditions, suggesting that oxygen molecules participate in the inhibition. Oxygen consumption measurements showed that the luteolin-7-G/iron ion complexes react with oxygen molecules in competition with 13-HPODE acid, and free iron ions exclusively react with 13-HPODE acid. The reaction of luteolin-7-G/iron ion complexes with oxygen molecules possibly diminishes the formation of pentyl and 7-carboxyheptyl radicals.

Isolation and characterization of Arabidopsis thaliana ISU1 gene.

We describe the isolation of a cDNA encoding Arabidopsis thaliana ISU1 (AtISU1), which regulates iron homeostasis in the mitochondria. The AtISU1 gene contained an open reading frame that encoded 167 amino acid residues. Northern blot analysis demonstrated that AtISU1 gene was ubiquitously expressed in plant tissues examined. The yeast seo5-1, which harbors a single base-pair deletion in ScISU1, is a suppressor of oxidative damage in sod1-deficient mutant. Based on comparative expression analyses using yeast ISU1 gene (ScISU1) in seo5-1 mutant, we found that AtISU1 acts as a counterpart of ScISU1.

Nob1p is required for biogenesis of the 26S proteasome and degraded upon its maturation in Saccharomyces cerevisiae.

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 ump1Delta cells, and temperature-sensitive nob1 and pno1 mutants exhibited defects in the processing of the beta 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 ump1Delta 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.