Assessing hydrophobic regions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae.
The hydrophobic, photoactivatable probe TID [3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine] was used to label the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The H(+)-ATPase accounted for 43% of the total label associated with plasma membrane protein and incorporated 0.3 mol of [125I]TID per mol of 100 kDa polypeptide. The H(+)-ATPase was purified by octyl glucoside extraction and glycerol gradient centrifugation, and was cleaved by either cyanogen bromide digestion or limited tryptic proteolysis to isolate labeled fragments. Cyanogen bromide digestion resulted in numerous labeled fragments of mass less than 21 kDa. Seven fragments suitable for microsequence analysis were obtained by electrotransfer to poly(vinylidene difluoride) membranes. Five different regions of amino-acid sequence were identified, including fragments predicted to encompass both membrane-spanning and cytoplasmic protein structure domains. Most of the labeling of the cytoplasmic domain was concentrated in a region comprising amino acids 347 to 529. This catalytic region contains the site of phosphorylation and was previously suggested to be hydrophobic in character (Goffeau, A. and De Meis, L. (1990) J. Biol. 265, 15503-15505). Complementary labeling information was obtained from an analysis of limited tryptic fragments enriched for hydrophobic character. Six principal labeled fragments, of 29.6, 20.6, 16, 13.1, 11.4 and 9.7 kDa, were obtained. These fragments were found to comprise most of the putative transmembrane region and a portion of the cytoplasmic region that overlapped with the highly labeled active site-containing cyanogen bromide fragment. Overall, the extensive labeling of protein structure domains known to lie outside the bilayer suggests that [125I]TID labeling patterns cannot be unambiguously interpreted for the purpose of discerning membrane-embedded protein structure domains. It is proposed that caution should be applied in the interpretation of [125I]TID labeling patterns of the yeast plasma membrane H(+)-ATPase and that new and diverse approaches should be developed to provide a more definitive topology model.
Seto-Young
D
D
Public Health Research Institute, New York, NY 10016.
Monk
B C
BC
Perlin
D S
DS
D000595:Amino Acid Sequence; D001389:Azirines; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D003488:Cyanogen Bromide; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin
D001389:Azirines; D010446:Peptide Fragments; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D014357:Trypsin; D006180:Proton-Translocating ATPases; D003488:Cyanogen Bromide