THE MEMBRANE TOPOLOGY OF THE RAT SARCOPLASMIC AND ENDOPLASMIC-RETICULUM CALCIUM ATPASES BY IN-VITRO TRANSLATION SCANNING

The membrane topology of the rat endoplasmic reticulum (ER) and sarcop lasmic reticulum (SR) Ca2+ ATPases were investigated using in vitro tr anscription/translation of fusion vectors containing DNA sequences enc oding putative membrane-spanning domains. The sequences of these Ca2ATPases are identical except for the COOH-terminal end, which contains an additional predicted transmembrane segment in the ER ATPase. The M O and M1 fusion vectors (Bamberg, K., and Sachs, G. (1994) J. Biol. Ch em. 269, 16909-16919) encode the NH2-terminal 101 (MO vector) or 139 ( M1 vector) amino acids of the H,K-ATPase alpha subunit followed by a l inker region for insertion of putative transmembrane sequences and, fi nally, the COOH-terminal 177 amino acids of the H,K-ATPase beta subuni t containing five N-linked glycosylation consensus sequences. The link er region was replaced by the putative transmembrane domains of the Ca 2+ ATPases, either individually or in pairs. Transcription and transla tion were performed using [S-35]methionine in a reticulocyte lysate sy stem in the absence or presence of canine pancreatic microsomes. The t ranslated fusion protein was identified by autoradiography following s eparation using SDS-polyacrylamide gel electrophoresis. When testing s ingle transmembrane segments, this method detects signal anchor activi ty with MO or stop transfer activity with M1. The first four predicted SERCA transmembrane domains acted as both signal anchor and stop tran sfer sequences. A construct containing the fifth predicted transmembra ne segment was able to act only as a stop transfer sequence. The sixth transmembrane segment did not insert cotranslationally into the membr ane. The seventh was able to act as both a signal anchor and stop tran sfer sequence, and the eighth showed stop transfer ability in the M1 v ector. The ninth transmembrane segment had both signal anchor and stop transfer capacity, whereas the tenth transmembrane segment showed onl y stop transfer sequence properties. The eleventh transmembrane sequen ce, unique to the ER Ca2+ ATPase, had both signal anchor and stop tran sfer properties. These translation data provide direct experimental ev idence for 8 or 9 of the 10 or 11 predicted transmembrane sequences in the current topological models for the SR or ER Ca2+ ATPases, respect ively.