Functional properties of a hybrid vacuolar H(+)-ATPase in Saccharomyces cells expressing the Nephrops 16-kDa proteolipid.
The hydrophobic 16-kDa polypeptide which forms gap-junction-like structures in the crustacean Nephrops norvegicus is a member of a highly conserved family of proteolipids involved in a variety of membrane transport functions in eukaryotic cells. This family also includes the product of the Saccharomyces cerevisiae VMA3 gene which encodes an integral membrane component of the vacuolar membrane H(+)-ATPase. The cDNA for the Nephrops proteolipid complements a mutation in the yeast VMA3 gene, resulting in assembly of a hybrid H(+)-ATPase comprising yeast catalytic subunits and Nephrops integral membrane components. The hybrid vacuolar ATPase was capable of ATP hydrolysis which was coupled to proton translocation and showed inhibitor binding and enzymological properties similar to those of wild-type V-ATPases (Km for ATP, 0.4 mM), suggesting that both yeast and crustacean proteolipids share conserved structure at regions of protein interaction. To facilitate isolation of the Nephrops proteolipid by affinity chromatography on a Ni(2+)-binding support, six C-terminal histidine residues were added to the proteolipid. This modification did not prohibit assembly into the hybrid H(+)-ATPase, although the resultant enzyme did have a markedly elevated Km (1.8 mM). The membrane-bound Vo sector of the ATPase was isolated by the affinity-chromatography procedure and reconstituted into synthetic vesicles. This complex was found to be impermeable to small cations in the absence of catalytic ATPase subunits either in situ in the vacuolar membrane or in the reconstituted system. The functional significance of this impermeability and the structure/function relationships between proteolipids from different sources are discussed.
Harrison
M A
MA
Department of Biochemistry and Molecular Biology, University of Leeds, England.
Jones
P C
PC
Kim
Y I
YI
Finbow
M E
ME
Findlay
J B
JB
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003445:Crustacea / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009532:Nickel; D009571:Nitrilotriacetic Acid; D055503:Protein Multimerization; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011522:Protons; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014617:Vacuoles / Q000201:enzymology*; D014638:Vanadates / Q000494:pharmacology
D011510:Proteolipids; D011522:Protons; D011994:Recombinant Proteins; C087027:proteolipid, Nephrops; D014638:Vanadates; D009532:Nickel; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009571:Nitrilotriacetic Acid