Cytoplasmic location of amino acids 359–440 of the Neurospora crassa plasma membrane H +-ATPase

Abstract

The topographic location of the region comprising amino acids 359–440 of the Neurospora crassa plasma membrane H +-ATPase has been clucidated using reconstituted proteoliposomes and protein chemical techniques. Proteoliposomes containing H +-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were cleaved with trypsin and the resulting digest was subjected to centrifugation on a glycerol step gradient to separate the released and liposome-bound peptides. The released peptides were recovered in the upper regions of the step gradient, whereas the liposome-bound peptides were recovered near the 40% glycerol interface. The released peptides present in the upper fractions were reduced, 14C-car☐ylmethylated, and then separated by high performance liquid chromatography. Two radioactive cysteine-containing peptides with retention times of about 162 and 182 min were identified as H +-ATPase peptides comprising residues Leu 363-Lys 379 and Leu 388-Arg 414, respectively, by comparison to standards prepared from the purified ATPase. This information thus establishes a cytoplasmic location for residues 359–418 in the H +-ATPase polypeptide chain. It also infers a cytoplasmic location for residues 419–440, since this stretch of amino acids is too short to cross the membrane and return between regions known to be cytoplasmically located. These results and the results of other recent experiments establish the topographical location of nearly all of the 919 residues in the H +-ATPase molecule.