Four hydrophobic segments in the NH2-terminal third (H1-H4) of Na,K-ATPase alpha subunit alternately initiate and halt membrane translocation of the newly synthesized polypeptide.

Transmembrane disposition of the NH2-terminal third of the Na,K-ATPase alpha subunit was studied using an experimental approach that involved in vitro endoplasmic reticulum membrane insertion of chimeras. These chimeras consisted of four truncated amino-terminal segments of the alpha subunit linked at amino acid residues 126, 179, 313, and 439 to chloramphenicol acetyltransferase (CAT), a reporter protein, that contains a consensus sequence for N-linked glycosylation. The fusion sites were located after one of the four hydrophobic segments (H1-H4). The results showed that the chimeras in which the alpha subunit was truncated at positions 126 and 313 were glycosylated, and the glycosylated peptides were protected by membranes from proteolysis. However, the other two chimeras were not glycosylated and the inserted peptides were digested by protease into fragments which did not immunoprecipitate with anti-CAT. These results clearly demonstrate that hydrophobic segments H1 and H3 function as signal/anchor type II, and H2 and H4 function as halt transfer signals. Furthermore, membrane insertion of the NH2-terminal third of Na,K-ATPase alpha subunit is achieved by a series of alternate signal/anchor type II and halt transfer sequences.