The Proteolipid of the A1A0ATP Synthase from Methanococcus jannaschii Has Six Predicted Transmembrane Helices but Only Two Proton-translocating Carboxyl Groups

doi:10.1074/jbc.274.36.25281

The Proteolipid of the A₁A₀ATP Synthase from Methanococcus jannaschii Has Six Predicted Transmembrane Helices but Only Two Proton-translocating Carboxyl Groups*

From the ^‡Lehrstuhl für Mikrobiologie der Ludwig-Maximilians-Universität München, Maria-Ward-Strasse 1a, 80638 München, Germany, ^§FB5, AG Mikrobiologie, Universität Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany, ^¶Max-Planck-Institut für Biochemie, Abteilung Proteinchemie, Am Klopferspitz 18a, 82152 Martinsried, Germany, and ^‖Lehrstuhl für Mikrobiologie, Universitätsstrasse 31, Universität Regensburg, 93053 Regensburg, Germany

Abstract

The proteolipid, a hydrophobic ATPase subunit essential for ion translocation, was purified from membranes ofMethanococcus jannaschii by chloroform/methanol extraction and gel chromatography and was studied using molecular and biochemical techniques. Its apparent molecular mass as determined in SDS-polyacrylamide gel electrophoresis varied considerably with the conditions applied. The N-terminal sequence analysis made it possible to define the open reading frame and revealed that the gene is a triplication of the gene present in bacteria. In some of the proteolipids, the N-terminal methionine is excised. Consequently, two forms with molecular masses of 21,316 and 21,183 Da were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The molecular and biochemical data gave clear evidence that the mature proteolipid from M. jannaschii is a triplication of the 8-kDa proteolipid present in bacterial F₁F₀ ATPases and most archaeal A₁A₀ ATPases. Moreover, the triplicated form lacks a proton-translocating carboxyl group in the first of three pairs of transmembrane helices. This finding puts in question the current view of the evolution of H⁺ ATPases and has important mechanistic consequences for the structure and function of H⁺ ATPases in general.

Footnotes

↵* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to V. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of Holger W. Jannasch, a pioneer of deep sea microbiology.
↵** To whom correspondence should be addressed. Tel.: 49-89-21806126; Fax: 49-89-21806127; E-mail: v.mueller@lrz.uni-muenchen.de.
Abbreviations:

DCCD

N,N′-dicyclohexylcarbodiimide

MALDI-TOF MS

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

ΔG_p

phosphorylation potential

Δp

proton-motive force or electrochemical proton potential

PAGE

polyacrylamide gel electrophoresis

bp

base pair(s)

CHAPS

3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

MES

4-morpholineethanesulfonic acid

TES

2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid
- Received April 6, 1999.
- Revision received June 21, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.