CtpA,a Copper-translocating P-type ATPase Involved in the Biogenesis of Multiple Copper-requiring Enzymes

The ctpA (ccoI) gene product, a putative inner membrane copper-translocating P1B-type ATPase present in many bacteria, has been shown to be involved only in the cbb₃ assembly in Rhodobacter capsulatus and Bradyrhizobium japonicum. ctpA was disrupted in Rubrivivax gelatinosus, and the mutants showed a drastic decrease in both cbb₃ and caa₃ oxidase activities. Inactivation of ctpA results also in a decrease in the amount of the nitrous oxide reductase, NosZ. This pleiotropic phenotype could be partially rescued by excess copper in the medium, indicating that CtpA is likely a copper transporter that supplies copper-requiring proteins in the membrane with this metal. Although CtpA shares significant sequence homologies with the homeostasis copper efflux P1B-type ATPases including the bacterial CopA and the human ATP7A and ATP7B, disruption of ctpA did not result in any sensitivity to excess copper. This indicates that the CtpA is not crucial for copper tolerance but is involved in the assembly of membrane and periplasmic copper enzymes in this bacterium. The potential roles of CtpA in bacteria in comparison with CopA are discussed.     

The Linker Region Plays a Regulatory Role in Assembly and Activity of the Vps4 AAA ATPase

The AAA-type ATPase Vps4 functions with components of the ESCRT (endosomal sorting complex required for transport) machinery in membrane fission events that are essential for endosomal maturation, cytokinesis, and the formation of retroviruses. A key step in these events is the assembly of monomeric Vps4 into the active ATPase complex, which is aided in part by binding of Vps4 via its N-terminal MIT (microtubule interacting and trafficking) domain to its substrate ESCRT-III. We found that the 40-amino acid linker region between the MIT and the ATPase domain of Vps4 is not required for proper function but plays a role in regulating Vps4 assembly and ATPase activity. Deletion of the linker is expected to bring the MIT domains into close proximity to the central pore of the Vps4 complex. We propose that this localization of the MIT domain in linker-deleted Vps4 mimics a repositioning of the MIT domain normally caused by binding of Vps4 to ESCRT-III. This structure would allow the Vps4 complex to engage ESCRT-III subunits with both the pore and the MIT domain simultaneously, which might be essential for the ATP-driven disassembly of ESCRT-III.     

The Mitochondrial Quality Control Protein Yme1 Is Necessary to Prevent Defective Mitophagy in a Yeast Model of Barth Syndrome

The Saccharomyces cerevisiae TAZ1 gene is an orthologue of human TAZ; both encode the protein tafazzin. Tafazzin is a transacylase that transfers acyl chains with unsaturated fatty acids from phospholipids to monolysocardiolipin to generate cardiolipin with unsaturated fatty acids. Mutations in human TAZ cause Barth syndrome, a fatal childhood cardiomyopathy biochemically characterized by reduced cardiolipin mass and increased monolysocardiolipin levels. To uncover cellular processes that require tafazzin to maintain cell health, we performed a synthetic genetic array screen using taz1Δ yeast cells to identify genes whose deletion aggravated its fitness. The synthetic genetic array screen uncovered several mitochondrial cellular processes that require tafazzin. Focusing on the i-AAA protease Yme1, a mitochondrial quality control protein that degrades misfolded proteins, we determined that in cells lacking both Yme1 and Taz1 function, there were substantive mitochondrial ultrastructural defects, ineffective superoxide scavenging, and a severe defect in mitophagy. We identify an important role for the mitochondrial protease Yme1 in the ability of cells that lack tafazzin function to maintain mitochondrial structural integrity and mitochondrial quality control and to undergo mitophagy.     

TORC2 Is Required to Maintain Genome Stability during S Phase in Fission Yeast

DNA damage can occur due to environmental insults or intrinsic metabolic processes and is a major threat to genome stability. The DNA damage response is composed of a series of well coordinated cellular processes that include activation of the DNA damage checkpoint, transient cell cycle arrest, DNA damage repair, and reentry into the cell cycle. Here we demonstrate that mutant cells defective for TOR complex 2 (TORC2) or the downstream AGC-like kinase, Gad8, are highly sensitive to chronic replication stress but are insensitive to ionizing radiation. We show that in response to replication stress, TORC2 is dispensable for Chk1-mediated cell cycle arrest but is required for the return to cell cycle progression. Rad52 is a DNA repair and recombination protein that forms foci at DNA damage sites and stalled replication forks. TORC2 mutant cells show increased spontaneous nuclear Rad52 foci, particularly during S phase, suggesting that TORC2 protects cells from DNA damage that occurs during normal DNA replication. Consistently, the viability of TORC2-Gad8 mutant cells is dependent on the presence of the homologous recombination pathway and other proteins that are required for replication restart following fork replication stalling. Our findings indicate that TORC2 is required for genome integrity. This may be relevant for the growing amount of evidence implicating TORC2 in cancer development.     

YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae

NAD⁺ is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD⁺ pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD⁺ metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD⁺ levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD⁺ homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD⁺ metabolism in higher eukaryotes.     

CgCYN1, a plasma membrane cystine-specific transporter of Candida glabrata with orthologues prevalent among pathogenic yeast and fungi

We describe a novel plasma membrane cystine transporter, CgCYN1, from Candida glabrata, the first such transporter to be described from yeast and fungi. C. glabrata met15Δ strains, organic sulfur auxotrophs, were observed to utilize cystine as a sulfur source, and this phenotype was exploited in the discovery of CgCYN1. Heterologous expression of CgCYN1 in Saccharomyces cerevisiae met15Δ strains conferred the ability of S. cerevisiae strains to grow on cystine. Deletion of the CgCYN1 ORF (CAGL0M00154g) in C. glabrata met15Δ strains caused abrogation of growth on cystine with growth being restored when CgCYN1 was reintroduced. The CgCYN1 protein belongs to the amino acid permease family of transporters, with no similarity to known plasma membrane cystine transporters of bacteria and humans, or lysosomal cystine transporters of humans/yeast. Kinetic studies revealed a K(m) of 18 ± 5 μM for cystine. Cystine uptake was inhibited by cystine, but not by other amino acids, including cysteine. The structurally similar cystathionine, lanthionine, and selenocystine alone inhibited transport, confirming that the transporter was specific for cystine. CgCYN1 localized to the plasma membrane and transport was energy-dependent. Functional orthologues could be demonstrated from other pathogenic yeast like Candida albicans and Histoplasma capsulatum, but were absent in Schizosaccharomyces pombe and S. cerevisiae.     

Evidence That the Yeast Desaturase Ole1p Exists as a Dimer in Vivo

Desaturase enzymes are composed of two classes, the structurally well characterized soluble class found predominantly in the plastids of higher plants and the more widely distributed but poorly structurally defined integral membrane class. Despite their distinct evolutionary origins, the two classes both require an iron cofactor and molecular oxygen for activity and are inhibited by azide and cyanide, suggesting strong mechanistic similarities. The fact that the soluble desaturase is active as a homodimer prompted us test the hypothesis that an archetypal integral membrane desaturase from Saccharomyces cerevisiae, the Δ⁹-acyl-Co-A desaturase Ole1p, also exhibits a dimeric organization. Ole1p was chosen because it is one of the best characterized integral membrane desaturase and because it retains activity when fused with epitope tags. FLAG-Ole1p was detected by Western blotting of immunoprecipitates in which anti-Myc antibodies were used for capture from yeast extracts co-expressing Ole1p-Myc and Ole1p-FLAG. Interaction was confirmed by two independent bimolecular complementation assays (i.e. the split ubiquitin system and the split luciferase system). Co-expression of active and inactive Ole1p subunits resulted in an ∼75% suppression of the accumulation of palmitoleic acid, demonstrating that the physiologically active form of Ole1p in vivo is the dimer in which both protomers must be functional.     

Regulatory Role of Proteasome in Determination of Platelet Life Span

Limit of platelet life span (8–10 days) is determined by the activity of a putative “internal clock” composed of Bcl-2 family proteins, whereas the role of other molecular players in this process remains obscure. Here, we sought to establish a central role of proteasome in platelet life span regulation. Administration of mice with inhibitors of proteasome peptidase activity induced significant thrombocytopenia. This was associated with enhanced clearance of biotin-labeled platelets from circulation and reduction in average platelet half-life from 66 to 37 h. Cells pretreated in vitro with proteasome inhibitors exhibited augmented annexin V binding and a drop in mitochondrial transmembrane potential indicative of apoptotic cell death and decreased platelet life span. These cells were preferentially phagocytosed by monocyte-derived macrophages, thus linking proteasome activity with platelet survival. The decisive role of proteasome in this process was underscored from enhanced expression of conformationally active Bax in platelets with attenuated proteasome activity, which was consistent with pro-apoptotic phenotype of these cells. The present study establishes a critical role of proteasome in delimiting platelet life span ostensibly through constitutive elimination of the conformationally active Bax. These findings bear potential implications in clinical settings where proteasome peptidase activities are therapeutically targeted.     

ATP-dependent Pre-replicative Complex Assembly Is Facilitated by Adk1p in Budding Yeast

Pre-replicative complex (pre-RC) assembly is a critical part of the mechanism that controls the initiation of DNA replication, and ATP binding and hydrolysis by multiple pre-RC proteins are essential for pre-RC assembly and activation. Here, we demonstrate that Adk1p (adenylate kinase 1 protein) plays an important role in pre-RC assembly in Saccharomyces cerevisiae. Isolated from a genetic screen, adk1^G20S cells with a mutation within the nucleotide-binding site were defective in replication initiation. adk1Δ cells were viable at 25 °C but not at 37°C. Flow cytometry indicated that both the adk1-td (temperature-inducible degron) and adk1^G20S mutants were defective in S phase entry. Furthermore, Adk1p bound to chromatin throughout the cell cycle and physically interacted with Orc3p, whereas the Adk1^G20S protein had a reduced ability to bind chromatin and Orc3p without affecting the cellular ATP level. In addition, Adk1p associated with replication origins by ChIP assay. Finally, Adk1-td protein depletion prevented pre-RC assembly during the M-to-G₁ transition. We suggest that Adk1p regulates ATP metabolism on pre-RC proteins to promote pre-RC assembly and activation.     

Discovery of a novel site regulating glucokinase activity following characterization of a new mutation causing hyperinsulinemic hypoglycemia in humans

Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S(0.5), 3.3 ± 0.1 and 3.5 ± 0.1 mm, respectively) versus wild type (7.5 ± 0.1 mm). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (<0.1). T103I had an altered affinity for glucose (S(0.5), 24.9 ± 0.6 mm), whereas V389D also exhibited a reduced affinity for ATP and decreased catalysis rate (S(0.5), 78.6 ± 4.5 mm; ATP(K(m)), 1.5 ± 0.1 mm; K(cat), 10.3 ± 1.1s(-1)) compared with wild type (ATP(K(m)), 0.4 ± <0.1; K(cat), 62.9 ± 1.2). Both Thr-103 mutants showed reduced inhibition by the endogenous hepatic inhibitor glucokinase regulatory protein. Molecular modeling demonstrated that Thr-103 maps to the allosteric activator site, whereas Val-389 is located remotely to this position and all other previously reported activating mutations, highlighting α-helix 11 as a novel region regulating GCK activity. Our data suggest that pharmacological manipulation of GCK activity at locations distal from the allosteric activator site is possible.     

The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication

The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1⁺, two temperature-sensitive mcb1 gene mutants (mcb1^ts) were isolated. Extensive genetic analysis showed that the mcb1^ts mutants were suppressed by a mcm5⁺ multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1^ts mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1^ts mutants. Furthermore, the mcb1^ts mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex.     

Loss of Vacuolar H+-ATPase Activity in Organelles Signals Ubiquitination and Endocytosis of the Yeast Plasma Membrane Proton pump Pma1p

Yeast mutants lacking the intracellular V-ATPase proton pump (vma mutants) have reduced levels of the Pma1p proton pump at the plasma membrane and increased levels in organelles including the vacuolar lumen. We examined the mechanism and physiological consequences of Pma1p mislocalization. Pma1p is ubiquitinated in vma mutants, and ubiquitination depends on the ubiquitin ligase Rsp5p and the arrestin-related adaptor protein Rim8p. vma mutant strains containing rsp5 or rim8 mutations maintain Pma1p at the plasma membrane, suggesting that ubiquitination is required for Pma1p internalization. Acute inhibition of V-ATPase activity with concanamycin A triggers Pma1p ubiquitination and internalization. In an endocytosis-deficient mutant (end4Δ) Pma1p is ubiquitinated but retained at the plasma membrane during concanamycin A treatment. Consistent with specificity in signaling loss of V-ATPase activity to Pma1p, another plasma membrane transporter, Mup1p, is not internalized in a vma mutant, and loss of the Mup1p adaptor Art1p does not prevent Pma1p internalization in a vma mutant. Very poor growth of vma2 rsp5-1 and vma2 rim8Δ double mutants suggests that Pma1p internalization benefits the vma mutants. We hypothesize that loss of V-ATPase-mediated organelle acidification signals ubiquitination, internalization, and degradation of a portion of Pma1p as a means of balancing overall pH homeostasis.     

腺甘酸环化酶Cyr1突变对酿酒酵母时序寿命的影响

为了研究腺甘酸环化酶Cyr1对酵母细胞时序寿命的影响,我们使用Cyr1△与野生菌DBY746来检测酵母细胞的时序寿命,H_2O_2或热抵抗能力以及线粒体膜形态。结果表明,Cyr1△的时序寿命是DBY746的2.25倍,在强卡路里限制调节下,两者的生存时间均有延长,DBY746和Cyr1△达到10%生存率的时间分别为13d和45d。Cyr1△有更强的抵抗H_2O_2和热处理能力,同时发现Cyr1△比DBY746在培养第3d和第5d有更完整的线粒体膜形态。     

Homocysteine Methyltransferases Mht1 and Sam4 Prevent the Accumulation of Age-damaged (R,S)-AdoMet in the Yeast Saccharomyces cerevisiae

The biological methyl donor S-adenosyl-l-methionine (AdoMet) is spontaneously degraded by inversion of its sulfonium center to form the R,S diastereomer. Unlike its precursor, (S,S)-AdoMet, (R,S)-AdoMet has no known cellular function and may have some toxicity. Although the rate of (R,S)-AdoMet formation under physiological conditions is significant, it has not been detected at substantial levels in vivo in a wide range of organisms. These observations imply that there are mechanisms that either dispose of (R,S)-AdoMet or convert it back to (S,S)-AdoMet. Previously, we identified two homocysteine methyltransferases (Mht1 and Sam4) in yeast capable of recognizing and metabolizing (R,S)-AdoMet. We found similar activities in worms, plants, and flies. However, it was not established whether these activities could prevent R,S accumulation. In this work, we show that both the Mht1 and Sam4 enzymes are capable of preventing R,S accumulation in Saccharomyces cerevisiae grown to stationary phase; deletion of both genes results in significant (R,S)-AdoMet accumulation. To our knowledge, this is the first time that such an accumulation of (R,S)-AdoMet has been reported in any organism. We show that yeast cells can take up (R,S)-AdoMet from the medium using the same transporter (Sam3) used to import (S,S)-AdoMet. Our results suggest that yeast cells have evolved efficient mechanisms not only for dealing with the spontaneous intracellular generation of the (R,S)-AdoMet degradation product but for utilizing environmental sources as a nutrient.     

Autoinhibitory regulation of TrwK, an essential VirB4 ATPase in type IV secretion systems

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. In a previous work we demonstrated that TrwK is able to hydrolyze ATP. Here, based on the structural homology of VirB4 proteins with the DNA-pumping ATPase TrwB coupling protein, we generated a series of variants of TrwK where fragments of the C-terminal domain were sequentially truncated. Surprisingly, the in vitro ATPase activity of these TrwK variants was much higher than that of the wild-type enzyme. Moreover, addition of a synthetic peptide containing the amino acid residues comprising this C-terminal region resulted in the specific inhibition of the TrwK variants lacking such domain. These results indicate that the C-terminal end of TrwK plays an important regulatory role in the functioning of the T4SS.