Topological analysis of H+,K(+)-ATPase using in vitro translation

Abstract

The membrane topology of the alpha subunit of the H+,K(+)-ATPase was investigated by using in vitro transcription/translation of DNA sequences encoding fusion proteins that contained possible membrane-spanning segments. The vectors consisted of DNA sequences encoding (a) either the first 101 (M0 vectors) or the first 139 (M1 vectors) amino acids of the N-terminal region of the alpha subunit of the ATPase, (b) a variable region, and then (c) the C-terminal 177 amino acids of the C-terminal region of the beta subunit, with five N-linked glycosylation sites. The variable region of the fusion protein contained the cDNA sequences representing the possible eight or 10 membrane-spanning segments either alone or in various combinations. Transcription/translation was performed in the presence of [35S]methionine using a coupled reticulocyte lysate in the absence and presence of microsomes. The fusion protein was identified by autoradiography following separation using SDS-polyacrylamide gel electrophoresis. Glycosylation of a translated sequence corresponded to membrane insertion and translocation of the C-terminal beta sequence. This method allowed analysis of signal anchor sequences using the M0 vector. The presence of a stop transfer sequence in the variable segment of the M1 vector resulted in inhibition of translocation of the C-terminal beta sequence. The sequences for the first four membrane segments could act as either signal anchor or stop transfer sequences. Therefore, this region of the alpha subunit has four membrane-spanning segments that are co-inserted with translation. The sequence corresponding to membrane segment M8 acted as a stop transfer sequence. The sequence corresponding to membrane segment M9 acted as a signal anchor sequence, and that corresponding to membrane segment M10 acted as a stop transfer sequence. The sequences representing the fifth, sixth, and seventh (M5, M6, and M7) membrane segments were unable to co-insert into the membrane. These data verify the first four and the eight membrane-spanning segments of the alpha subunit of the gastric H+,K(+)-ATPase and provide evidence for translational insertion of an additional pair of membrane-spanning segments, M9 and M10. It appears that insertion of membrane segments M5, M6, and M7 is determined differently from the other membrane-spanning segments. In combination with other methods, this in vitro transcription/translation method is useful for defining the membrane topology of the P type ATPases.