Abstract
The proton-translocating A1A0 ATP synthase/hydrolase of Methanosarcina mazei Gö1 was purified and shown to consist of six subunits of molecular masses of 65, 49, 40, 36, 25, and 7 kDa. Electron microscopy revealed that this enzyme is organized in two domains, the hydrophilic A1 and the hydrophobic A0 domain, which are connected by a stalk. Genes coding for seven hydrophilic subunits were cloned and sequenced. From these data it is evident that the 65-, 49-, 40- and 25-kDa subunits are encoded by ahaA, ahaB, ahaC, and ahaD, respectively; they are part of the A1 domain or the stalk. In addition there are three more genes, ahaE, ahaF, and ahaG, encoding hydrophilic subunits, which were apparently lost during the purification of the protein. The A0 domain consists of at least the 7-kDa proteolipid and the 36-kDa subunit for which the genes have not yet been found. In summary, it is proposed that the A1A0 ATPase of Methanosarcina mazei Gö1 contains at least nine subunits, of which seven are located in A1 and/or the stalk and two in A0.
Highlights
Methanogenesis from H2 ϩ CO2 as catalyzed by the nonmarine methanogenic Archaea Methanosarcina barkeri or Methanosarcina mazei Go1 is obligatorily coupled to the generation of two primary ion gradients at the same time: the reduction of the heterodisulfide of coenzyme M1 and 7-mercaptoheptanoylthreoninephosphate is coupled to electrogenic translocation of protons across the membrane; in addition the penultimate step of methanogenesis, the transfer of the methyl group from methyltetrahydromethanopterin to coenzyme M as catalyzed by the corrinoid-containing multi-subunit enzyme methyltetrahydromethanopterin:coenzyme M methyltransferase, is coupled to vectorial sodium ion translocation across the membrane [1]
The archaeal A1A0 ATPase2 shares properties with both, bacterial F1F0 and eucaryal V1V0 ATPases [3, 4]. It clearly functions as an ATP synthase, which is in accordance with F1F0 but in sharp contrast to V1V0 ATPases; the structure of the proteolipid, which is in the range of 6 – 8 kDa in A1A0 and F1F0 ATPases but 16 kDa in V1V0 ATPases, was suggested to be at least one of the reasons for this difference [5,6,7,8]
Since M. mazei Go1 was recently shown by determining rates of ATP synthesis under various conditions to contain a Naϩtranslocating F1F0 as well as a Hϩ-translocating A1A0 ATP synthase [2], it was essential to analyze the membrane-bound ATPase activity in more detail
Summary
Materials—All chemicals were reagent grade and were purchased form Merck AG, Darmstadt, Germany. Purification of the A1A0 ATPase—Cells were harvested in the mid-log phase, washed and suspended in membrane buffer (Tris-HCl, pH 8.0, 20 mM NaHSO3, 5 mM MgSO4, 40 mM KCl, 0.2 mM dithiothreitol, 0.2 mM EGTA, 10% (v/v) glycerol, 1 M phenylmethylsulfonyl fluoride, 6 mM p-aminobenzamidine) and disrupted in a French pressure cell at a pressure of 138 MPa. Debris was removed by centrifugation (25,000 ϫ g, 10 min). Molecular Procedures—Chromosomal DNA of M. mazei Go1 was isolated as described [28], restricted, size-fractionated by gradient centrifugation and cloned into either pSE420 or pGem-7Zf-(ϩ). Southern blot analysis of genomic DNA of M. mazei Go1 restricted with different enzymes revealed a 11-kbp XbaI fragment that hybridized with the homologous probe This fragment was cloned into pGem-7Zf-(ϩ) and the recombinant plasmid was named pCF1. DNA sequence was determined by the chain termination method of Sanger and analyzed on a VAX computer using the GCG package [29, 30]
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