The first putative transmembrane helix of the 16 kDa proteolipid lines a pore in the Vo sector of the vacuolar H(+)-ATPase.
The 16 kDa proteolipid is the major component of the vacuolar H(+)-ATPase membrane sector, responsible for proton translocation. Expression of a related proteolipid from the arythropod Nephrops norvegicus in a Saccharomyces strain in which the VMA3 gene for the endogenous proteolipid has been disrupted results in restored vacuolar H(+)-ATPase function. We have used this complementation system, coupled to cysteine substitution mutagenesis and protein chemistry, to investigate structural features of the proteolipid. Consecutive cysteines were introduced individually into putative transmembrane segment 1 of the proteolipid, and at selected sites in extramembranous regions and in segment 3 and 4. Analysis of restored vacuolar H(+)-ATPase function showed that segment 1 residues sensitive to mutation to cysteine were clustered on a single face, but only if the segment was helical. Only residues insensitive to mutation could be covalently modified by the cysteine-specific reagent fluorescein 5-maleimide. A cysteine introduced into segment 3 was the only residue accessible to a relatively hydrophobic reagent, suggesting accessibility to the lipid phase. Analysis of disulphide bond formation between introduced cysteines indicates that the first transmembrane alpha-helices of each monomer are adjacent to each other at the centre of the proteolipid multimeric complex. The data are consistent with a model in which the fluorescein maleimide-accessible face of helix I lines a pore at the centre of a hexameric complex formed by the proteolipid, with the mutationally sensitive face oriented into the protein core. The implications for ion-transport function in this family of proteins are discussed in the context of this structural model.
Jones
P C
PC
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Harrison
M A
MA
Kim
Y I
YI
Finbow
M E
ME
Findlay
J B
JB
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005452:Fluoresceins / Q000737:chemistry; D015151:Immunoblotting; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D008121:Nephropidae / Q000235:genetics; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000737:chemistry* / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D014617:Vacuoles / Q000201:enzymology*
D004220:Disulfides; D005452:Fluoresceins; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D011510:Proteolipids; C037041:fluorescein 5-maleimide; D006180:Proton-Translocating ATPases; D003545:Cysteine