P4 ATPases - The physiological relevance of lipid flipping transporters

P4 ATPases are integral transmembrane proteins implicated in phospholipid translocation from the exoplasmic to the cytosolic leaflet of biological membranes. Our present knowledge on the cellular physiology of P4 ATPases is mostly derived from studies in the yeast Saccharomyces cerevisiae, where P4 ATPases play a pivotal role in the biogenesis of intracellular transport vesicles, polarized protein transport and protein maturation. In contrast, the physiological and cellular functions of mammalian P4 ATPases are largely unexplored. P4 ATPases act in concert with members of the CDC50 protein family, which are putative β-subunits for P4 ATPases. This review highlights the current status of a slowly emerging research field and emphasizes the contribution of P4 ATPases to the vesicle-generating machinery.     

Identification and phylogenetic classification of eleven putative P-type calcium transport ATPase genes in the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe

Calcium is an essential second messenger in yeast metabolism and physiology. So far, only four genes coding for calcium translocating ATPases had been discovered in yeast. The recent completion of the yeastSaccharomyces cerevisiae genome allowed us to identify six new putative Ca⁺⁺-ATPases encoding genes. Protein sequence homology analysis and phylogenetic classification of all putative Ca⁺⁺-ATPase gene products from the yeastsSaccharomyces cerevisiae andSchizosacchraomyces pombe reveal three clusters of homologous proteins. Two of them comprises seven proteins which might belong to a new class of P-type ATPases of unknown subcellular location and of unknown physiological function.     

Cloning and Characterization of an ATPase Gene from Pneumocystis carinii which Closely Resembles Fungal H+ ATPases

ABSTRACT. A gene encoding a P-type cation translocating ATPase was cloned from a genomic library of rat-derived Pneumocystis carinii. The nucleotide sequence of the gene contains a 2781 base-pair open reading frame that is predicted to encode a 101, 401 dalton protein composed of 927 amino acids. The P. carinii ATPase protein (pcal) is 69–75% identical when compared with eight proton pumps from six fungal species. The Pneumocystis ATPase is less than 34% identical to ATPase proteins from protozoans, vertebrates or the Ca⁺⁺ ATPases of yeast. The P. carinii ATPase contains 115 of 121 residues previously identified as characteristic of H⁺ ATPases. Alignment of the Pneumocystis and fungal proton pumps reveals five homologous domains specific for fungal H⁺ ATPases.     

Homology of ATP binding sites from Ca2+ and (Na,K)-ATPases: comparison of the amino acid sequences of fluorescein isothiocyanate labeled peptides

Ca2+ and (Na,K)-stimulated ATPases from various species and tissues were labeled with fluorescein isothiocyanate (FITC). Labeled peptides were solubilized by tryptic digestion and purified by reverse phase high pressure liquid chromatography. The amino acid sequences of the labeled peptides reveal considerable homology between sarcoplasmic reticulum Ca2+-ATPases from various sources. These Ca2+-ATPases also contain a region of homology with all other ATPases thus far sequenced. A difference was demonstrated between dog skeletal and cardiac Ca2+-ATPases. These results demonstrate homology of the putative ATP binding site of ATPases, which extends over tissue, species, and cation specificity, including the completely conserved amino acid sequence: lys-gly-ala-pro-glu.     

Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry

The 20S proteasome functions in protein degradation in eukaryotes together with the 19S ATPases or in archaea with the homologous PAN ATPase complex. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X motif (HbYX). We show that these residues are essential for PAN to associate with the 20S and open its gated channel for substrate entry. Upon ATP binding, these C-terminal residues bind to pockets between the 20S's alpha subunits. Seven-residue or longer peptides from PAN's C terminus containing the HbYX motif also bind to these sites and induce gate opening in the 20S. Gate opening could be induced by C-terminal peptides from the 19S ATPase subunits, Rpt2, and Rpt5, but not by ones from PA28/26, which lack the HbYX motif and cause gate opening by distinct mechanisms. C-terminal residues in the 19S ATPases were also shown to be critical for gating and stability of 26S proteasomes. Thus, the C termini of the proteasomal ATPases function like a "key in a lock" to induce gate opening and allow substrate entry.     

Evolutionary primacy of sodium bioenergetics

Background  

The F- and V-type ATPases are rotary molecular machines that couple translocation of protons or sodium ions across the membrane to the synthesis or hydrolysis of ATP. Both the F-type (found in most bacteria and eukaryotic mitochondria and chloroplasts) and V-type (found in archaea, some bacteria, and eukaryotic vacuoles) ATPases can translocate either protons or sodium ions. The prevalent proton-dependent ATPases are generally viewed as the primary form of the enzyme whereas the sodium-translocating ATPases of some prokaryotes are usually construed as an exotic adaptation to survival in extreme environments.     

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.     

The contribution of mouse models to our understanding of systemic candidiasis

To maintain optimal intracellular concentrations of alkali-metal-cations, yeast cells use a series of influx and efflux systems. Nonconventional yeast species have at least three different types of efficient transporters that ensure potassium uptake and accumulation in cells. Most of them have Trk uniporters and Hak K(+)-H(+) symporters and a few yeast species also have the rare K(+) (Na(+))-uptake ATPase Acu. To eliminate surplus potassium or toxic sodium cations, various yeast species use highly conserved Nha Na(+) (K(+))/H(+) antiporters and Na(+) (K(+))-efflux Ena ATPases. The potassium-specific yeast Tok1 channel is also highly conserved among various yeast species and its activity is important for the regulation of plasma membrane potential.     

F- and V-type ATPases in the hyperthermophilic bacterium Thermotoga neapolitana

Two gene clusters encoding F- or V-type ATPases were found in genomic DNA of the hyperthermophilic bacterium Thermotoga neapolitana. The subunit genes of each ATPase formed an operon. While the gene arrangement in the operon of the F-type ATPase resembled those in eukaryotic organelles and bacteria, that of the V-type ATPase was different from those reported for archaea, bacteria, or eukaryotes. Both ATPases were found to be expressed in the cells of T. neapolitana by Western blot analysis. Although V-type ATPase could not be rendered soluble, F-type ATPase was solubilized with 1% Triton X-100 and characterized. This is the first report of the coexistence of both F- and V-type ATPases in hyperthermophilic bacteria. It has recently been shown by a genome analysis that Thermotoga maritima has no V-type ATPase gene cluster but does have an F-type ATPase gene cluster; however, part of a gene for the D-subunit of the V-type ATPase gene has been reported in the T. maritima genome. Evolution of the two types of ATPases in Thermotoga is discussed.     

Conversion of energy in halobacteria: ATP synthesis and phototaxis

Halobacteria are aerobic chemo-organotroph archaea that grow optimally between pH 8 and 9 using a wide range of carbon sources. These archaea have developed alternative processes of energy provision for conditions of high cell densities and the reduced solubility of molecular oxygen in concentrated brines. The halobacteria can switch to anaerobic metabolism by using an alternative final acceptor in the respiratory chain or by fermentation, or alternatively, they can employ photophosphorylation. Light energy is converted by several retinal-containing membrane proteins that, in addition to generating a proton gradient across the cell membrane, also make phototaxis possible in order to approach optimal light conditions. The structural and functional features of ATP synthesis in archaea are discussed, and similarities to F-ATPases (functional aspects) or vacuolar ATPases (structural aspects) are presented. Received: 18 December 1995 / Accepted: 3 April 1996     

Structure of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum as studied by electron tomography.

Valosine-containing protein-like ATPase from Thermoplasma acidophilum is a member of the superfamily of ATPases associated with a diversity of cellular activities and is closely related to CDC48 from yeast and p97 from higher eukaryotes and more distantly to N-ethylmaleimide-sensitive fusion protein. We have used electron tomography to obtain low-resolution (2-2.5 nm) three-dimensional maps of both the whole 500 kDa complex and the N-terminally truncated valosine-containing protein-like ATPase from T. acidophilum complex lacking the putative substrate binding domain.     

The Archaeal Proteasome Is Regulated by a Network of AAA ATPases

The proteasome is the central machinery for targeted protein degradation in archaea, Actinobacteria, and eukaryotes. In its basic form, it consists of a regulatory ATPase complex and a proteolytic core particle. The interaction between the two is governed by an HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) at the C terminus of the ATPase subunits, which stimulates gate opening of the proteasomal α-subunits. In archaea, the proteasome-interacting motif is not only found in canonical proteasome-activating nucleotidases of the PAN/ARC/Rpt group, which are absent in major archaeal lineages, but also in proteins of the CDC48/p97/VAT and AMA groups, suggesting a regulatory network of proteasomal ATPases. Indeed, Thermoplasma acidophilum, which lacks PAN, encodes one CDC48 protein that interacts with the 20S proteasome and activates the degradation of model substrates. In contrast, Methanosarcina mazei contains seven AAA proteins, five of which, both PAN proteins, two out of three CDC48 proteins, and the AMA protein, function as proteasomal gatekeepers. The prevalent presence of multiple, distinct proteasomal ATPases in archaea thus results in a network of regulatory ATPases that may widen the substrate spectrum of proteasomal protein degradation.     

Purirication and characterization of two forms of DNA-dependent ATPase from yeast

Two forms of DNA-dependent ATPase activity have been purified from yeast extracts. The two ATPases differ from each other in chromatographic properties and heat stabilities but have similar molecular weight and reaction properties. DNA-dependent ATPase I has been purified to near homogeneity, while DNA-dependent ATPase II is only partially purified. The two ATPases from yeast are related structurally since antiserum raised against ATPase I cross-react against ATPase II. Yeast DNA-dependent ATPase I has a native molecular weight of about 68,000 and consists of a single polypeptide chain. ATPase II also sediments on sucrose gradient as a 68,000-dalton protein. Both yeast DNA-dependent ATPases hydrolyze dNTPs and rNTPs to their corresponding nucleoside diphosphates and orthophosphate, but dATP and ATP are preferred substrates. In addition to nucleoside triphosphates, both enzymes require a divalent cation and a polynucleotide for activity. Single-stranded DNAs and polydeoxynucleotides are the most effective co-substrates for yeast DNA-dependent ATPases. Addition of yeast DNA-dependent ATPases to DNA synthesis system containing yeast DNA polymerases does not significantly stimulate the rate of DNA synthesis.     

嗜盐古菌Haloferax sp. D1227中超氧化物歧化酶功能鉴定及其增强细菌耐盐性的研究

【背景】细菌、酵母或植物来源的超氧化物歧化酶(Superoxide dismutase,SOD)编码基因在异源宿主中表达并提高宿主耐盐性的研究已有一些报道,其异源宿主也多为植物,而古菌来源的超氧化物歧化酶编码基因在细菌中成功表达并提高其耐盐性的研究尚无报道。【目的】寻找嗜盐古菌Haloferax sp.D1227中的超氧化物歧化酶编码基因并鉴定其功能,将其在4-硝基苯酚降解细菌Burkholderia sp.SJ98中表达,研究该古菌的超氧化物歧化酶对菌株SJ98耐盐性和降解4-硝基苯酚功能的影响。【方法】通过生物信息学方法寻找嗜盐古菌D1227中潜在的超氧化物歧化酶编码基因,利用表达载体p ET-28a和广泛宿主载体p BBR1MCS-2将其分别在E.coli BL21(DE3)和4-硝基苯酚的降解菌株SJ98中异源表达,检测细胞抽提液和纯化蛋白的超氧化物歧化酶比活力。分别以葡萄糖和4-硝基苯酚为碳源,在M9培养基和添加500 mmol/L Na Cl(Na Cl含量约3%)的M9培养基中分别培养细菌SJ98的重组菌株和空载体重组菌株,利用全自动生长曲线分析仪和高效液相色谱等方法检测重组菌株的生长能力和对4-硝基苯酚的降解能力。【结果】通过生物信息学分析,在嗜盐古菌D1227基因组中发现了潜在的超氧化物歧化酶编码基因sod A,其在E.coli BL21(DE3)和菌株SJ98中分别异源表达均具有超氧化物歧化酶活力[细胞抽提液的比活力分别为21.07±0.02 U/mg和84.56±0.16 U/mg,从BL21(DE3)菌株纯化的蛋白Sod AD1227比活力为179.46±3.43 U/mg]。在添加500 mmol/L Na Cl的M9培养基中培养时,以葡萄糖为碳源,重组菌株SJ98[p BBR-sod A]仍可正常生长,而空载体对照菌株SJ98[p BBR1MCS-2]几乎丧失了生长能力;以4-硝基苯酚为碳源,菌株SJ98[p BBR-sod A]保持了利用底物生长和降解底物的能力,而菌株SJ98[p BBR1MCS-2]的生长和降解能力几乎丧失。用软件Phyre2模拟分析Sod AD1227的单体结构,该蛋白拥有Fe/Mn-SOD家族的典型结构特征,推测其属于Fe/Mn-SOD家族。【结论】本研究为利用古菌SOD对细菌进行改造以适应高盐环境中降解有机污染物的应用提供了潜在的可行性。     

Assembly of the regulatory complex of the 26S proteasome

The 19S regulatory complex (RC) of 26S proteasomes is a 900–1000 kDa particle composed of 18 distinct subunits (S1–S15) ranging in molecular mass from 25 to 110 kDa. This particle confers ATP-dependence and polyubiquitin (polyUb) recognition to the 26S proteasome. The symmetry and homogenous structure of the proteasome contrasts sharply with the remarkable complexity of the RC. Despite the fact that the primary sequences of all the subunits are now known, insight has been gained into the function of only eight subunits. The six ATPases within the RC constitute a subfamily (S4-like ATPases) within the AAA superfamily and we have shown that they form specific pairs in vitro[1]. We have now determined that putative coiled-coils within the variable N-terminal regions of these proteins are likely to function as recognition elements that direct the proper placement of the ATPases within the RC. We have also begun mapping putative interactions between non-ATPase subunits and S4-like ATPases. These studies have allowed us to build a model for the specific arrangement of 9 subunits within the human regulatory complex. This model agrees with recent findings by Glickman et al. [2] who have reported that two subcomplexes, termed the base and the lid, form the RC of budding yeast 26S proteasomes.     

Inhibition of the proton pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide

The inhibition of the proton-pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide (DCCD) can be correlated with the covalent incorporation of the inhibitor. Full inhibition of the yeast enzyme required the incorporation of about 1 mol DCCD/mol of the ATPase polypeptide of 100 kDa. A kinetic study of the interaction of DCCD with the yeast and oat ATPases indicates a second-order rate constant of about 500 M-1 min-1 and a stoichiometry of 1 mol DCCD/mol of enzyme, in agreement with the amount of DCCD incorporated by the yeast enzyme. It is proposed that DCCD reacts with a single carboxylic group present in a hydrophobic region of these proton-pumping ATPases and which could participate in proton binding and transport.     

The evolution of the ribosome biogenesis pathway from a yeast perspective

Ribosome biogenesis is fundamental for cellular life, but surprisingly little is known about the underlying pathway. In eukaryotes a comprehensive collection of experimentally verified ribosome biogenesis factors (RBFs) exists only for Saccharomyces cerevisiae. Far less is known for other fungi, animals or plants, and insights are even more limited for archaea. Starting from 255 yeast RBFs, we integrated ortholog searches, domain architecture comparisons and, in part, manual curation to investigate the inventories of RBF candidates in 261 eukaryotes, 26 archaea and 57 bacteria. The resulting phylogenetic profiles reveal the evolutionary ancestry of the yeast pathway. The oldest core comprising 20 RBF lineages dates back to the last universal common ancestor, while the youngest 20 factors are confined to the Saccharomycotina. On this basis, we outline similarities and differences of ribosome biogenesis across contemporary species. Archaea, so far a rather uncharted domain, possess 38 well-supported RBF candidates of which some are known to form functional sub-complexes in yeast. This provides initial evidence that ribosome biogenesis in eukaryotes and archaea follows similar principles. Within eukaryotes, RBF repertoires vary considerably. A comparison of yeast and human reveals that lineage-specific adaptation via RBF exclusion and addition characterizes the evolution of this ancient pathway.