Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase.
Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.
Duarte
Afonso M S
AM
Laboratory of Biophysics, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
Wolfs
Cor J A M
CJ
van Nuland
Nico A J
NA
Harrison
Michael A
MA
Findlay
John B C
JB
van Mierlo
Carlo P M
CP
Hemminga
Marcus A
MA
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*
D008823:Micelles; D010446:Peptide Fragments; D011522:Protons; D025262:Vacuolar Proton-Translocating ATPases