Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding.
The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. Much less clear is the fate of membrane-embedded molecular machines in the gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, we can link specific lipid and nucleotide binding with distinct regulatory roles.
Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ.
Min
M
Zhou
Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ.
Nina
N
Morgner
Department of Chemistry, Lensfield Road, University of Cambridge CB2 1EW.|Department of Physiology, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile.
Nelson P
NP
Barrera
Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ.
Argyris
A
Politis
Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ.
Shoshanna C
SC
Isaacson
Department of Chemistry, Lensfield Road, University of Cambridge CB2 1EW.
Dijana
D
Matak-Vinković
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
Takeshi
T
Murata
Department of Chemistry, University of Texas at El Paso, El Paso, Texas 79968, USA.
Ricardo A
RA
Bernal
The Victor Chang Cardiac Research Institute, Lowy Packer Building, 405 Liverpool Street, Darlinghurst NSW 2010.|Faculty of Medicine, University of New South Wales, Sydney 2052, Australia.
Daniela
D
Stock
Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ.
Carol V
CV
Robinson
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002308:Cardiolipins / Q000032:analysis / Q000378:metabolism; D016983:Enterococcus / Q000201:enzymology; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D008563:Membrane Lipids / Q000032:analysis / Q000378:metabolism*; D008958:Models, Molecular; D010714:Phosphatidylethanolamines / Q000032:analysis / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D016963:Thermus thermophilus / Q000201:enzymology*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D002308:Cardiolipins; D008563:Membrane Lipids; D010714:Phosphatidylethanolamines; D021122:Protein Subunits; C483858:phosphatidylethanolamine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C500332:V-type sodium ATPase, Enterococcus hirae; D025262:Vacuolar Proton-Translocating ATPases