Structure, function and regulation of the vacuolar (H)-ATPases

The vacuolar (H⁺)-ATPases (or V-ATPases) function to acidify intracellular compartments in eukaryotic cells, playing an important role in such processes as receptor-mediated endocytosis, intracellular membrane traffic, protein degradation and coupled transport. V-ATPases in the plasma membrane of specialized cells also function in renal acidification, bone resorption and cytosolic pH maintenance. The V-ATPases are composed of two domains. The V₁ domain is a 570-kDa peripheral complex composed of 8 subunits (subunits A–H) of molecular weight 70–13 kDa which is responsible for ATP hydrolysis. The V₀ domain is a 260-kDa integral complex composed of 5 subunits (subunits a–d) which is responsible for proton translocation. The V-ATPases are structurally related to the F-ATPases which function in ATP synthesis. Biochemical and mutational studies have begun to reveal the function of individual subunits and residues in V-ATPase activity. A central question in this field is the mechanism of regulation of vacuolar acidification in vivo. Evidence has been obtained suggesting a number of possible mechanisms of regulating V-ATPase activity, including reversible dissociation of V₁ and V₀ domains, disulfide bond formation at the catalytic site and differential targeting of V-ATPases. Control of anion conductance may also function to regulate vacuolar pH. Because of the diversity of functions of V-ATPases, cells most likely employ multiple mechanisms for controlling their activity.     

A Spin System Labeled and Highly Resolved ed-H(CCO)NH-TOCSY Experiment for the Facilitated Assignment of Proton Side Chains in Partially Deuterated Samples

The introduction of deuterated and partially deuterated protein samples has greatly facilitated the 13C assignment of larger proteins. Here we present a new version of the HC(CO)NH-TOCSY experiment, the ed-H(CCO)NH-TOCSY experiment for partially deuterated samples, introducing a multi-quantum proton evolution period. This approach removes the main relaxation source (the dipolar coupling to the directly bound 13C spin) and leads to a significant reduction of the proton and carbon relaxation rates. Thus, the indirect proton dimension can be acquired with high resolution, combined with a phase labeling of the proton resonances according to the C-C spin system topology. This editing scheme, independent of the CHn multiplicity, allows to distinguish between proton side-chain positions occurring within a narrow chemical shift range. Therefore this new experiment facilitates the assignment of the proton chemical shifts of partially deuterated samples even of high molecular weights, as demonstrated on a 31 kDa protein.     

Solvent Exchange Rates of Side-chain Amide Protons in Proteins

Solvent exchange rates and temperature coefficients for Asn/Gln side-chain amide protons have been measured in Escherichia coli HPr. The protons of the eight side-chain amide groups (two Asn and six Gln) exhibit varying exchange rates which are slower than some of the fast exchanging backbone amide protons. Differences in exchange rates of the E and Z protons of the same side-chain amide group are obtained by measuring exchange rates at pH values > 8. An NOE between a side-chain amide proton and a bound water molecule was also observed.     

应用离子注入技术对家蚕形态学及遗传学进行的初步研究(英文)

离子注入技术是将某种元素的原子进行电离,并使其在电场中加速,在获得较高的速度后射入固体材料表面。在离子注入过程中,被电离的离子在电场作用下加速运动,离子靠着本身获得的动能进入基体表面,在表层中运动的离子与基体原子作用损失能量后在一定的位置停留下来。该技术自６０年代问世以来,主要用于材料改性等方面。８０年代中期,我国学者开始将其用于农作物育种方面的研究,大大拓宽了离子注入技术的应用领域。所用实验材料的基因及表现型见Ｔａｂ３,我们将氢离子（Ｅ＝３５ＭｅＶ）注入处于胚胎发育后期的家蚕卵内（Ｔａｂ１）,观察其对家蚕形态及遗传方面的影响,结果表明：（１）在家蚕胚胎发育的已４期注入氢离子,其半致死剂量ＬＤ５０为１ｘ１０１０～１ｘ１０１１ｃｍ２这一区间之内;当剂量达到１ｘ１０１２ｃｍ２时,已全部致死（Ｆｉｇ．１＆Ｔａｂ．２）;（２）注入氢离子能够使家蚕在第１腹节上产生褐斑（Ｆｉｇ．２）的频率增高。并首次观察到因注入氢离子而导致家蚕出现非成对的褐斑（Ｆｉｇ．３＆Ｔａｂ．４）。（３）在氢离子注入剂量为１ｘ１０１０ｃｍ２时,能够诱变产生大量的嵌合体家蚕,并且诱变频率高达３８．５％（Ｆｉｇ．４＆Ｔａｂ．５）,这样高的     

Protonmotive Mechanism of Heme-Copper Oxidases

The mechanism of coupling of proton and electron transfer in oxidases is reviewed and related to the structural information that is now available. A glutamate trap mechanism for proton/electron coupling is described.     

ATP-synthesis in archaea: Structure-function relations of the halobacterial A-ATPase

The archaeal (A)-ATPase has been described as a chimeric energy converter with close relationship to both the vacuolar ATPase class in higher eukaryotes and the coupling factor (F)-ATPase class in eubacteria, mitochondria and chloroplasts. With respect to their structure and some inhibitor responses, A-ATPases are more closely related to the vacuolar ATPase type than to F-ATPase. Their function, ATP synthesis at the expense of an ion gradient, however, is a typical attribute of the F-ATPase class. V-type ATPases serve as generators of a proton gradient driving the accumulation of solutes within vesicles such as the vacuoles of plant cells. The three catalytic subunits (A) of the archaeal ATPases are the largest subunits of the A1-part and, like in V-ATPases, closer related to the F-ATPase -subunits, whereas B corresponds to F-ATPase . The catalytic subunits A of archaeal ATPases contain an insert of about 80 amino acids in their primary structures that may be aligned to comparable structures in V-ATPases. The location of this additional peptide in Haloferax volcanii is shown using the 2.8 Å X-ray resolution of the bovine mitochondrial F-ATPase [Abrahams et al. (1994) Nature 370: 621-628]. A three dimensional structure for the catalytic subunit of Haloferax volcanii ATPase is proposed using the Swiss-Model Automated Protein Modelling Server. The halobacterial ATPase is a halophilic protein; it contains about 20% negatively charged amino acid residues. A large portion of acidic residues is located on the outer surface of the protein as well as in the insert of subunit A. This result is discussed in terms of protein stability under high salt stress conditions.     

Human carbonic anhydrase I: Effect of specific site mutations on its function

Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. X-ray crystallographic and inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in other CA isoenzymes identified several residues, in particular Thr199, GlulO6, Tyr7, Glull7, His l07, with likely involvement in the catalytic activity of HCAI. To further study the role of these residues, we undertook, site-directed mutagenesis of HCAI. Using a polymerase chain reaction based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes encoding proteins with single amino acid substitutions. Thrl99Val, Thrl99Cys, Thr199Ser, GlulO6Ile, Glul06Gln, Tyr7Trp, Glu.117Gln, and His 107Val mutations were thus generated and the activity of each measured by ester hydrolysis. Overproduction of the Glu117Gln and HisI07Val mutant proteins inEscherichia coli resulted in a large proportion of the enzyme forming aggregates probably due to folding defect. The mutations Thr199Val, GlulO6Ile and GlulO6Gln gave soluble protein with drastically reduced enzyme activity, while the Tyr7Trp mutation had only marginal effect on the activity, thus s.uggesting important roles for Thr199 and Glu lO6 but not for Tyr7 in the catalytic function of HCAI.     

The Na transport in gram-positive bacteria defect in the Mrp antiporter complex measured with Na nuclear magnetic resonance

²³Na nuclear magnetic resonance (NMR) has previously been used to monitor Na⁺ translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na⁺. In this work, the ²³Na NMR method was adapted for measurements of internal Na⁺ concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na⁺ translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na⁺ concentration than an external one.     

S6K1 is a multifaceted regulator of Mdm2 that connects nutrient status and DNA damage response

p53 mediates DNA damage‐induced cell‐cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR‐S6K1 through p38α MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2‐mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR‐S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53‐dependent cell death. These findings thus establish mTOR‐S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1–Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging‐controlling Mdm2–p53 and mTOR‐S6K pathways.