Protein misfolding caused by exposure to arsenite is associated with transcriptional activation of the AIRAP gene. We report here that AIRAP is an arsenite-inducible subunit of the proteasome's 19S cap that binds near PSMD2 at the 19S base. Compared to the wild-type, knockout mouse cells or C. elegans lacking AIRAP accumulate more polyubiquitylated proteins and exhibit higher levels of stress when exposed to arsenite, and proteasomes isolated from arsenite-treated AIRAP knockout cells are relatively impaired in substrate degradation in vitro. AIRAP's association with the 19S cap reverses the stabilizing affect of ATP on the 26S proteasome during particle purification, and AIRAP-containing proteasomes, though constituted of 19S and 20S subunits, acquire features of hybrid proteasomes with both 19S and 11S regulatory caps. These features include enhanced cleavage of peptide substrates and suggest that AIRAP adapts the cell's core protein degradation machinery to counteract proteotoxicity induced by an environmental toxin.

The Ubiquitin Proteasome System (UPS) was discovered in two steps. Initially, APF-1 (ATP-dependent proteolytic Factor 1) later identified as ubiquitin (Ub), a hitherto known protein of unknown function, was found to covalently modify proteins. This modification led to degradation of the tagged protein by - at that time - an unknown protease. This was followed later by the identification of the 26S proteasome complex which is composed of a previously identified Multi Catalytic Protease (MCP) and an additional regulatory complex, as the protease that degrades Ub-tagged proteins. While Ub conjugation and proteasomal degradation are viewed as a continued process responsible for most of the regulated proteolysis in the cell, the two processes have also independent roles. In parallel and in the years that followed, the hallmark signal that links the substrate to the proteasome was identified as an internal Lys48-based polyUb chain. However, since these initial findings were described, our understanding of both ends of the process (i.e. Ub-conjugation to proteins, and their recognition and degradation), have advanced significantly. This enabled us to start bridging the ends of this continuous process which suffered until lately from limited structural data regarding the 26S proteasomal architecture and the structure and diversity of the Ub chains. These missing pieces are of great importance because the link between ubiquitination and proteasomal processing is subject to numerous regulatory steps and are found to function improperly in several pathologies. Recently, the molecular architecture of the 26S proteasome was resolved in great detail, enabling us to address mechanistic questions regarding the various molecular events that polyubiquitinated (polyUb) substrates undergo during binding and processing by the 26S proteasome. In addition, advancement in analytical and synthetic methods enables us to better understand the structure and diversity of the degradation signal. The review summarizes these recent findings and addresses the extrapolated meanings in light of previous reports. Finally, it addresses some of the still remaining questions to be solved in order to obtain a continuous mechanistic view of the events that a substrate undergoes from its initial ubiquitination to proteasomal degradation. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Haploid yeast cells are capable of invading agar when grown on rich media. Cells of the ⌺1278b genetic background manifest this property, whereas other laboratory strains are incapable of invasive growth. We show that disruption of the RAS2 gene in the ⌺1278b background significantly reduces invasive growth but that expression of a constitutively active Ras2p (Ras2 Val19 p) in this strain has a minimal effect on its invasiveness. On the other hand, expression of Ras2 Val19 p in another laboratory strain, SP1, rendered it invasive. These results suggest that a hyperactive Ras2 pathway induces invasive growth and that this pathway might be overactive in the ⌺1278b genetic background. Indeed, cells of the ⌺1278b are defective in the induction of stress-responsive genes, while their Gcn4 target genes are constitutively transcribed. This pattern of gene expression was previously shown to be associated with an active Ras/cyclic AMP (cAMP) pathway. We show that suppression of stress-related genes in ⌺1278b cells is a result of their inability to activate transcription through the stress response element (STRE). Disruption of RAS2, which abolished invasiveness, induced an increase in STRE activity. Further, in the SP1 genetic background, disruption of either the MSN2/4 genes (encoding activators of STRE) or the yAP-1 gene was sufficient to restore invasive growth in ras2⌬ cells. We conclude that Ras2-mediated suppression of the stress response is sufficient to induce invasiveness. Accordingly, the fact that the stress response is suppressed in ⌺1278b background explains its invasiveness. It seems that invasiveness is a phenotype related to unregulated growth and is therefore manifested by cells harboring an overactive Ras/cAMP cascade. In this respect, invasiveness in yeast is reminiscent of the property of ras-transformed fibroblasts to invade soft agar.

The burden of protein misfolding is believed to contribute to aging. However, the links between adaptations to conditions associated with protein misfolding and resistance to the time-dependent attrition of cellular function remain poorly understood. We report that worms lacking aip-1, a homologue of mammalian AIRAP (arsenic-inducible proteasomal 19S regulatory particle-associated protein), are not only impaired in their ability to resist exposure to arsenite but also exhibit shortened lifespan and hypersensitivity to misfolding-prone proteins under normal laboratory conditions. Mammals have a second, constitutively expressed AIRAP-like gene (AIRAPL) that also encodes a proteasome-interacting protein, which shares with AIRAP the property of enhancing peptide accessibility to the proteasome's active site. Genetic rescue experiments suggest that features common to the constitutively expressed worm AIP-1 and mammalian AIRAPL (but missing in the smaller, arsenite-inducible AIRAP) are important to lifespan extension. In worms, a single AIRAP-related protein links proteasomal adaptation to environmental stress with resistance to both proteotoxic insults and maintenance of animal life span under normal conditions. aging ͉ chaperones ͉ environmental toxins ͉ protein degradation

SummaryIn an effort to understand how an accurate level of stress-specific expression is obtained, we studied the promoter of the yeast HSP104 gene. Through 5¢ deletions, we defined a 334 bp fragment upstream of the first coding AUG as sufficient and essential for maximal basal activity and a 260 bp fragment as sufficient and essential for heat shock responsiveness. These sequences contain heat shock elements (HSEs) and stress response elements (STREs) that cooperate to achieve maximal inducible expression. However, in the absence of one set of factors (e.g. in msn2Dmsn4D cells) proper induction is obtained exclusively through HSEs. We also show that HSP104 is constitutively derepressed in ras2D cells. This derepression is achieved exclusively through activation of STREs, with no role for HSEs. Strikingly, in ras2Dmsn2Dmsn4D cells the HSP104 promoter is also derepressed, but in this strain derepression is mediated through HSEs, showing the flexibility and adaptation of the promoter. Thus, appropriate transcription of HSP104 is usually obtained through cooperation between the Msn2/4/STRE and the HSF/ HSE systems, but each factor could activate the promoter alone, backing up the other. Transcription control of HSP104 is adaptive and robust, ensuring proper expression under extreme conditions and in various mutants. IntroductionIn order to survive under suboptimal conditions, cells have developed several types of responses, known as the cellular stress responses. A major stress response is the induction of expression (to a high level) of protective and repair systems, capable of combating the particular stress or damage. Another simultaneous response is the induc- of stresses ranging from oxidative and osmolar stress, to nitrogen starvation (Boy-Marcotte et al., 1998;Moskvina et al., 1998;Treger et al., 1998a). In addition, HSEs are found in the promoters of a limited number of stress genes (mainly encoding Hsps) whereas STREs are more abundant (Moskvina et al., 1998;Treger et al., 1998a). The STRE system could therefore be considered a general, non-specific stress response (Ruis and Schuller, 1995;Siderius and Mager, 1997).STREs serve as a binding site for transcriptional activators, containing Cys 2 His 2 zinc fingers. Two such factors, known as Msn2p and Msn4p, are activators of many STRE-containing genes (Martinez-Pastor et al., 1996;Schmitt and McEntee, 1996). Hot1p and Msn1p also activate some STRE-driven genes (Rep et al., 2000). A msn2Dmsn4D double mutant shows up to 10-fold reductions in the basal and induced expression of many stressrelated genes (Boy-Marcotte et al., 1996;Martinez-Pastor et al., 1996;Moskvina et al., 1998;Gasch et al., 2000;Causton et al., 2001). Notably, in spite of the central role of Msn2/4p in the stress response, expression of many stress genes is not totally abolished in msn2Dmsn4D cells (Treger et al., 1998b;Boy-Marcotte et al., 1999;Simon et al., 1999;Gasch et al., 2000;Rep et al., 2000). Furthermore, activation of genes such as HSP104, UBI4 and HSP78 is similar in wild-type and ...

The 26 S proteasome is the eukaryotic protease responsible for the degradation of most cellular proteins. As such it accommodates the ability to function under diverse conditions that the cell may encounter. This function is supported by various adaptors that modulate various aspects in protein degradation, these include regulation of substrate delivery, deubiquitination, unfolding, and 20 S gate dilation. Here we show a new functional complex between the P97 and the proteasome that is assembled in response to proteasomal impairment. This entails P97 binding to the 26 S proteasome via the 19 S particle thereby forming an additional hexameric ATPase ring to relieve repression. P97-bound proteasomes showed selective binding toward the Npl4-ufd1 P97 co-factors, indicating a unique cellular role for P97 binding to proteasomes. P97-bound proteasomes display enhanced activity, showing a relief in proteolysis impairment. Our findings place P97 directly in non-ERAD proteasomal functions and establish a new checkpoint in UPS impairment. The ability to modulate proteasome activity and properly respond to protein misfolding, is of great importance in cellular regulation.

Lys48-linked ubiquitin chains act as the main targeting signals for protein degradation by the proteasome. Here we report selective binding of AIRAPL, a protein that associates with the proteasome upon exposure to arsenite, to Lys48-linked tri-ubiquitin chains. AIRAPL comprises two ubiquitin-interacting motifs in tandem (tUIMs) that are linked through a flexible inter-UIM region. In the complex crystal structure UIM1 binds the proximal ubiquitin, whereas UIM2 (the double-sided UIM) binds non-symmetrically to the middle and distal ubiquitin moieties on either side of the helix. Specificity of AIRAPL for Lys48-linked ubiquitin chains is determined by UIM2, and the flexible inter-UIM linker increases avidity by placing the two UIMs in an orientation that facilitates binding of the third ubiquitin to UIM1. Unlike middle and proximal ubiquitins, distal ubiquitin binds UIM2 through a novel surface, which leaves the Ile44 hydrophobic patch accessible for binding to the proteasomal ubiquitin receptors.

Perturbation of the cytoplasmic protein folding environment by exposure to oxidative stress-inducing As(III)-containing compounds challenges the ubiquitin-proteasome system. Here we report on mass spectrometric analysis of As(III)-induced changes in the proteasome's composition in samples prepared by stable isotope labeling with amino acids in cell culture, using mammalian cells in which TRP32 (thioredoxin-related protein of 32 kDa; also referred to as TXNL1) was identified as a novel subunit of the 26 S proteasome. Quantitative genetic interaction mapping, using the epistatic miniarray profiling approach, identified a functional connection between TRP32 and the proteasome. Deletion of txl1, the Schizosaccharomyces pombe homolog of TRP32, results in a slow growth phenotype when combined with deletion of cut8, a gene required for normal proteasome localization. Deletion analysis in vivo, chemical crosslinking, and manipulation of the ATP concentration in vitro during proteasome immunopurification revealed that the C-terminal domain of mammalian TRP32 binds the 19 S regulatory particle in proximity to the proteasome substrate binding site. Thiol modification with polyethylene glycol-maleimide showed disulfide bond formation at the active site of TRP32 in cells exposed to As(III). Pulse-chase labeling showed that TRP32 is a stable protein whose half-life of >6 h is surprisingly reduced to 1 h upon exposure of cells to As(III). These findings reveal a previously undescribed thiol reductase at the proteasome's regulatory particle.