The proteasome 11S regulator subunit REG alpha (PA28 alpha) is a heptamer.

Activity of the 20S proteasome, which performs much of the cytosolic and nuclear proteolysis in eukaryotic cells, is controlled by regulatory complexes that bind to one or both ends of the cylindrical proteasome. One of these complexes, the 11S regulator (REG), is a complex of 28 kDa subunits that is thought to activate proteasomes toward the production of antigenic peptides. REG, purified from red blood cells, is a complex of REG alpha and REG beta subunits. We have crystallized recombinant REG alpha (rREG alpha) and collected diffraction data to 3.0 A resolution. The self-rotation function indicates that rREG alpha forms a heptameric ring in the crystal. Equilibrium sedimentation demonstrates that rREG alpha is a heptamer in solution also.     

The pertussis toxin S1 subunit is a thermally unstable protein susceptible to degradation by the 20S proteasome

Pertussis toxin (PT) is an AB-type protein toxin that consists of a catalytic A subunit (PT S1) and an oligomeric, cell-binding B subunit. It belongs to a subset of AB toxins that move from the cell surface to the endoplasmic reticulum (ER) before the A chain passes into the cytosol. Toxin translocation is thought to involve A chain unfolding in the ER and the quality control mechanism of ER-associated degradation (ERAD). The absence of lysine residues in PT S1 may allow the translocated toxin to avoid ubiquitin-dependent degradation by the 26S proteasome, which is the usual fate of exported ERAD substrates. As the conformation of PT S1 appears to play an important role in toxin translocation, we used biophysical and biochemical methods to examine the structural properties of PT S1. Our in vitro studies found that the isolated PT S1 subunit is a thermally unstable protein that can be degraded in a ubiquitin-independent fashion by the core 20S proteasome. The thermal denaturation of PT S1 was inhibited by its interaction with NAD, a donor molecule used by PT S1 for the ADP ribosylation of target G proteins. These observations support a model of intoxication in which toxin translocation, degradation, and activity are all influenced by the heat-labile nature of the isolated toxin A chain.     

The N terminus of the cardiac L-type Ca(2+) channel alpha(1C) subunit. The initial segment is ubiquitous and crucial for protein kinase C modulation, but is not directly phosphorylated.

The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca(2+) channel alpha(1C) subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating (Shistik, E., Ivanina, T., Blumenstein, Y., and Dascal, N. (1998) J. Biol. Chem. 273, 17901-17909). The mechanism of PKC action and the location of the PKC target site are not known. Moreover, uncertainties in the genomic sequence of the N-terminal region of alpha(1C) leave open the question of the presence of RH-type N terminus in L-type channels in mammalian tissues. Here, we demonstrate the presence of alpha(1C) protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of alpha(1C) expressed in Xenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathione S-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus.     

The yeast RNA1 protein, necessary for RNA processing, is homologous to the human ribonuclease/angiogenin inhibitor (RAI)

Summary Mutations in theRNA1 gene ofSaccharomyces cerevisiae, which encodes an essential cytosolic protein, affect the production and processing of all major classes of RNA. The mechanisms underlying these effects are not at all understood. Detailed comparative sequence analyses revealed that the RNA1 protein belongs to a superfamily, the members of which contain repetitive leucine-rich motifs (LRM). Within this superfamily RNA1 is most closely related to the ribonuclease/angiogenin inhibitor (RAI), which is a tightly binding inhibitor of ribonucleolytic activities in mammals. These results not only provide important clues to the structure, function and evolution of the RNAI protein, but also have intriguing implications for possible novel functions of RAI.     

The raised midpoint potential of the [2Fe2S] cluster of cytochrome bc1 is mediated by both the Qo site occupants and the head domain position of the Fe-S protein subunit

We have previously reported that mutant strains of Rhodobacter capsulatus that have alanine insertions (+nAla mutants) in the hinge region of the iron sulfur (Fe-S) containing subunit of the bc(1) complex have increased redox midpoint potentials (E(m)) for their [2Fe2S] clusters. The alteration of the E(m) in these strains, which contain mutations far from the metal binding site, implied that the local environment of the metal center is indirectly altered by a change in the interaction of this subunit with the hydroquinone oxidizing (Q(o)) site [Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) J. Biol. Chem. 277, 3464-3470]. Subsequently, the E(m) changes have been proposed to be predominantly due to a stronger or more stabilized hydrogen bonding between the reduced [2Fe2S] cluster and the Q(o) site inhabitant ubiquinone (Q) [Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002) Biochemistry 41, 14372-14382]. To further investigate this issue, Fe-S protein-Q interactions were monitored by electron paramagnetic resonance (EPR) spectroscopy and the findings indicated that the wild type and mutant proteins interactions with Q are similar. Moreover, when the Q(pool) was chemically depleted, the E(m) of the [2Fe2S] cluster in mutant bc(1) complexes remained more positive than a similarly treated native enzyme (e.g., the [2Fe2S] E(m) of the +2Ala mutant was 55 mV more positive than the wild type). These data suggest that the increased E(m) of the [2Fe2S] cluster in the +nAla mutants is in part due to the cluster's interaction with Q, and in part to additional factors that are independent of hydrogen bonding to Q. One such factor, the possibility of a different position of the Fe-S at the Q(o) site of the mutant proteins versus the native enzyme, was addressed by determining the orientation of the [2Fe2S] cluster in the membrane using EPR spectroscopy. In the case of the +2Ala mutant, the [2Fe2S] cluster orientation in the absence of inhibitor is different than that seen in the native enzyme. However, the +2Ala mutant cluster shared a similar orientation with the native enzyme when both samples were exposed to either stigmatellin or myxothiazol. In addition, Q(pool) extracted membranes of +2Ala mutant exhibited fewer overall orientations, with the predominant one being more similar to that observed in the non-Q-depleted membranes of the +2Ala mutant than the Q-depleted membranes of a wild-type strain. Therefore, additional component(s) that are independent of Q(o) site inhabitants and that originate from the newly observed orientations of the [2Fe2S] clusters in the +nAla mutants also contribute to the increased midpoint potentials of their [2Fe2S] clusters. While the molecular basis of these components remains to be determined, salient implications of these findings in terms of Q(o) site catalysis are discussed.     

The filamentation pathway controlled by the Efg1 regulator protein is required for normal biofilm formation and development in Candida albicans

Ralstonia eutropha JMP134 (pJP4) grows on 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4-D). The copy number of chlorocatechol genes has been observed to be important for allowing growth of bacterial strains on chloroaromatic compounds. Despite the fact that two functional chlorocatechol degradation tfd gene clusters are harbored on plasmid pJP4, a single copy of the region comprising all tfd genes in strain JMP134-F was insufficient to allow growth on 3-CB, whereas growth on 2,4-D was only slightly retarded compared to the wild-type strain. Using competitive PCR, approximately five copies of pJP4 per genome were observed to be present in the wild-type strain, whereas only one copy of pJP4 was present per chromosome in strain JMP134-F. Therefore, several copies of pJP4 per chromosome are required for full expression of the tfd-encoded growth abilities in the wild-type R. eutropha strain.     

Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity

[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.     

The interaction domain of the redox protein adrenodoxin is mandatory for binding of the electron acceptor CYP11A1, but is not required for binding of the electron donor adrenodoxin reductase

Adrenodoxin (Adx) is a [2Fe-2S] ferredoxin involved in electron transfer reactions in the steroid hormone biosynthesis of mammals. In this study, we deleted the sequence coding for the complete interaction domain in the Adx cDNA. The expressed recombinant protein consists of the amino acids 1-60, followed by the residues 89-128, and represents only the core domain of Adx (Adx-cd) but still incorporates the [2Fe-2S] cluster. Adx-cd accepts electrons from its natural redox partner, adrenodoxin reductase (AdR), and forms an individual complex with this NADPH-dependent flavoprotein. In contrast, formation of a complex with the natural electron acceptor, CYP11A1, as well as electron transfer to this steroid hydroxylase is prevented. By an electrostatic and van der Waals energy minimization procedure, complexes between AdR and Adx-cd have been proposed which have binding areas different from the native complex. Electron transport remains possible, despite longer electron transfer pathways.     

The transmembrane domain of the amyloid precursor protein is required for antiamyloidogenic processing by α-secretase ADAM10

Neurotoxic amyloid β-peptides are thought to be a causative agent of Alzheimer’s disease in humans. The production of amyloid β-peptides from amyloid precursor protein (APP) could be diminished by enhancing α-processing; however, the physical interactions between APP and α-secretases are not well understood. In this study, we employed super-resolution light microscopy to examine in cell-free plasma membranes the abundance and association of APP and α-secretases ADAM10 (a disintegrin and metalloproteinase) and ADAM17. We found that both secretase molecules localize similarly closely to APP (within ≤50 nm). However, when cross-linking APP with antibodies directed against the GFP tag of APP, in confocal microscopy, we observed that only ADAM10 coaggregated with APP. Furthermore, we mapped the involved protein domain by using APP variants with an exchanged transmembrane segment or lacking cytoplasmic/extracellular domains. We identified that the transmembrane domain of APP is required for association with α-secretases and, as analyzed by Western blot, for α-processing. We propose that the transmembrane domain of APP interacts either directly or indirectly with ADAM10, but not with ADAM17, explaining the dominant role of ADAM10 in α-processing of APP. Further understanding of this interaction may facilitate the development of a therapeutic strategy based on promoting APP cleavage by α-secretases.     

The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function

Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the β-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 °C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the β-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport.     

The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) protein is dispensable for DNA replication and the S phase checkpoint but required for the G2/M checkpoint

Budding yeast Dpb11 (human TopBP1, fission yeast Cut5) is an essential protein required for replisome assembly and for the DNA damage checkpoint. Previous studies with the temperature-sensitive dpb11-1 allele, truncated at amino acid 583 of the 764-amino acid protein, have suggested the model that Dpb11 couples DNA replication to the replication checkpoint. However, the dpb11-1 allele shows distinct replication defects even at permissive temperatures. Here, we determine that the 1-600-amino acid domain of DPB11 is both required and sufficient for full replication function of Dpb11 but that this domain is defective for activation of the principal checkpoint kinase Mec1 (human ataxia telangiectasia and Rad3-related) in vitro and in vivo. Remarkably, mutants of DPB11 that leave its replication function intact but abrogate its ability to activate Mec1 are proficient for the replication checkpoint, but they are compromised for the G(2)/M DNA damage checkpoint. These data suggest that replication checkpoint defects may result indirectly from defects in replisome assembly. Two conserved aromatic amino acids in the C terminus of Dpb11 are critical for Mec1 activation in vitro and for the G(2)/M checkpoint in yeast. Together with aromatic motifs identified previously in the Ddc1 subunit of 9-1-1, another activator of Mec1 kinase, they define a consensus structure for Mec1 activation.