The 26S proteasome, the central enzyme of the ubiquitin-proteasome system, is comprised of the 20S catalytic core particle (CP) and the 19S regulatory particle (RP), itself composed of two subcomplexes, the base and the lid. 20S proteasome assembly is assisted by several chaperones. Integral subunits of the RP participate in its assembly, but no external factors have been identified so far. Here we characterize the yeast Hsm3 protein, which displays unique features regarding 19S assembly. Hsm3 associates with 19S subcomplexes via a carboxy-terminal domain of the Rpt1 base subunit but is missing in the final 26S proteasome. Moreover, Hsm3 is specifically required for the base subcomplex assembly. Finally, we identify the putative species-specific 19S subunit S5b as a functional homolog of the Hsm3 chaperone in mammals. These findings shed light on chaperone-assisted proteasome assembly in eukaryotes.

R.Madrid and S.Le Maout contributed equally to this workAquaporin 4 (AQP4) is the predominant water channel in the brain. It is targeted to speci®c membrane domains of astrocytes and plays a crucial role in cerebral water balance in response to brain edema formation. AQP4 is also speci®cally expressed in the basolateral membranes of epithelial cells. However, the molecular mechanisms involved in its polarized targeting and membrane traf®cking remain largely unknown. Here, we show that two independent C-terminal signals determine AQP4 basolateral membrane targeting in epithelial MDCK cells. One signal involves a tyrosine-based motif; the other is encoded by a di-leucine-like motif. We found that the tyrosinebased basolateral sorting signal also determines AQP4 clathrin-dependent endocytosis through direct interaction with the m subunit of AP2 adaptor complex. Once endocytosed, a regulated switch in m subunit interaction changes AP2 adaptor association to AP3. We found that the stress-induced kinase casein kinase (CK)II phosphorylates the Ser276 immediately preceding the tyrosine motif, increasing AQP4±m3A interaction and enhancing AQP4±lysosomal targeting and degradation. AQP4 phosphorylation by CKII may thus provide a mechanism that regulates AQP4 cell surface expression. Keywords: adaptor protein complex/AQP4/ phosphorylation/protein sorting/regulated membrane traf®cking IntroductionAquaporin water channels are essential for mediating rapid osmotic water transport across cell membranes. The product of at least one of the 10 distinct mammalian aquaporin genes has been identi®ed in nearly all tissues, re¯ecting the fundamental nature of water transport processes. The ubiquitous character of aquaporins is associated with remarkably conserved structural features. Structural and functional analyses have revealed that aquaporins are homomultimeric complexes containing four identical subunits. All the aquaporin subunits identi®ed to date have N-and C-termini facing the cytosol, and contain six transmembrane domains that are connected by ®ve loops (Deen and van Os, 1998;Engel et al., 2000). Multiple aquaporins are often expressed in a single cell on different subcellular membrane domains where they are subjected to distinct regulatory cues. Although the phenomenon underlies a plethora of physiologically relevant water transport processes, little is known about the molecular mechanisms that govern the membrane targeting and expression of aquaporin channels.Consider, for example, the epithelial renal collecting duct principal cell and the cellular basis for the regulation of water balance. These cells express at least three different types of aquaporins in a polarized fashion. The vasopressin-regulated aquaporin 2 (AQP2) is speci®cally targeted to the apical cell domain. The hormone induces insertion of a vesicular pool of AQP2 channels in the apical membranes to allow ef®cient transcellular osmotic water re-absorption to occur in accord with physiological demands (Brown et al., 1988). The two other water channels, AQP3 ...

SummaryThe 2-cysteine peroxiredoxins (2-Cys-Prxs) are antioxidants that reduce peroxides through a thiol-based mechanism. During catalysis, these ubiquitous enzymes are occasionally inactivated by the substratedependent oxidation of the catalytic cysteine to the sulfinic acid (-SO 2 H) form, and are reactivated by reduction by sulfiredoxin (Srx), an enzyme recently identified in yeast and in mammal cells. In plants, 2-Cys-Prxs constitute the most abundant Prxs and are located in chloroplasts. Here we have characterized the unique Srx gene in Arabidopsis thaliana (AtSrx) from a functional point of view, and analyzed the phenotype of two AtSrx knockout (AtSrx-) mutant lines. AtSrx is a chloroplastic enzyme displaying sulfinic acid reductase activity, as shown by the ability of the recombinant AtSrx to reduce the overoxidized 2-Cys-Prx form in vitro, and by the accumulation of the overoxidized Prx in mutant lines lacking Srx in vivo. Furthermore, AtSrx mutants exhibit an increased tolerance to photooxidative stress generated by high light combined with low temperature. These data establish that, as in yeast and in mammals, plant 2-Cys-Prxs are subject to substrate-mediated inactivation reversed by Srx, and suggest that the 2-Cys-Prx redox status and sulfiredoxin are parts of a signaling mechanism participating in plant responses to oxidative stress.

The 26S proteasome, a molecular machine responsible for regulated protein degradation, consists of a proteolytic core particle (20S CP) associated with 19S regulatory particles (19S RPs) subdivided into base and lid subcomplexes. The assembly of 19S RP base subcomplex is mediated by multiple dedicated chaperones. Among these, Hsm3 is important for normal growth and directly targets the carboxyl-terminal (C-terminal) domain of Rpt1 of the Rpt1-Rpt2-Rpn1 assembly intermediate. Here, we report crystal structures of the yeast Hsm3 chaperone free and bound to the C-terminal domain of Rpt1. Unexpectedly, the structure of the complex suggests that within the Hsm3-Rpt1-Rpt2 module, Hsm3 also contacts Rpt2. We show that in both yeast and mammals, Hsm3 actually directly binds the AAA domain of Rpt2. The Hsm3 C-terminal region involved in this interaction is required in vivo for base assembly, although it is dispensable for binding Rpt1. Although Rpt1 and Rpt2 exhibit weak affinity for each other, Hsm3 unexpectedly acts as an essential matchmaker for the Rpt1-Rpt2-Rpn1 assembly by bridging both Rpt1 and Rpt2. In addition, we provide structural and biochemical evidence on how Hsm3/S5b may regulate the 19S RP association to the 20S CP proteasome. Our data point out the diverse functions of assembly chaperones.AAA ATPase | Arm/HEAT repeats | two-hybrid assay | native gel T he ubiquitin-proteasome system is a major proteolytic system in the cytosol and nucleus of all eukaryotic cells that regulates various essential cellular processes by degrading proteins, which, in most cases, are conjugated to ubiquitin (1-3). The 26S proteasome, the most downstream element of this pathway, is responsible for protein degradation. It comprises the catalytic core particle (20S CP) capped by one or two regulatory particles (19S RPs or PA700 in mammals), forming RP 1 CP and RP 2 CP complexes, respectively (4). The 20S CP encloses the protease active sites (5), whereas the 19S RP functions in substrate recognition, deubiquitination, unfolding, and translocation into the 20S CP and provides the ATP and ubiquitin dependence on the 20S CP (reviewed in 2).The 19S RP can be subdivided into two subcomplexes, namely, the base and the lid. The subunit architecture of the 19S RP has been further improved very recently (6, 7). The base contains six homologous ATPase subunits of the AAA family (referred to as Rpt1-6 in yeast) plus non-ATPase subunits: Rpn1, Rpn2, and Rpn13 (reviewed in 8). By analogy to proteasome-activating ATPase complexes in prokaryotes and archaeas, the 19S RP ATPases are presumed to assemble into a six-membered ring that directly abuts the 20S CP (reviewed in 8, 9). The Rpt1-Rpt5-Rpt4-Rpt3-Rpt6-Rpt2 arrangement within the ring, first proposed based on phylogenetic hypotheses (10), was recently experimentally confirmed (11). Rpt subunits consist of a coiled coil (CC) segment and an OB-fold domain, followed by an AAA ATPase domain (12, 13). The AAA ATPase domain comprises the C domain, which is a four-helix bundle. Protruding from the C...

Transcription and maintenance of genome integrity are fundamental cellular functions. Deregulation of transcription and defects in DNA repair lead to serious pathologies. The Mediator complex links RNA polymerase (Pol) II transcription and nucleotide excision repair via Rad2/XPG endonuclease. However, the functional interplay between Rad2/XPG, Mediator and Pol II remains to be determined. In this study, we investigated their functional dynamics using genomic and genetic approaches. In a mutant affected in Pol II phosphorylation leading to Mediator stabilization on core promoters, Rad2 genome-wide occupancy shifts towards core promoters following that of Mediator, but decreases on transcribed regions together with Pol II. Specific Mediator mutations increase UV sensitivity, reduce Rad2 recruitment to transcribed regions, lead to uncoupling of Rad2, Mediator and Pol II and to colethality with deletion of Rpb9 Pol II subunit involved in transcription-coupled repair. We provide new insights into the functional interplay between Rad2, Mediator and Pol II and propose that dynamic interactions with Mediator and Pol II are involved in Rad2 loading to the chromatin. Our work contributes to the understanding of the complex link between transcription and DNA repair machineries, dysfunction of which leads to severe diseases.

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Mediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts.

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