IN-VITRO TRANSLATION ANALYSIS OF INTEGRAL MEMBRANE-PROTEINS

A method of in vitro translation scanning was applied to a variety of polytopic integral membrane proteins, a transition metal P type ATPase from Helicobacter pylori, the SERCA 2 ATPase, the gastric H+,K+ ATPas e, the CCK-A receptor and the human heal bile acid transporter. This m ethod used vectors containing the N terminal region of the gastric H+, K+ ATPase or the N terminal region of the CCK-A receptor, coupled via a linker region to the last 177 amino acids of the beta-subunit of the gastric H+,K+ ATPase. The latter contains 5 potential N-Linked glycos ylation sites. Translation of vectors containing the cDNA encoding one , two or more putative transmembrane domains in the absence or presenc e of microsomes allowed determination of signal anchor or stop transfe r properties of the putative transmembrane domains by the molecular we ight shift on SDS PAGE. The P type ATPase from Helicobacter pylori sho wed the presence of 8 transmembrane segments with this method. The SER CA 2 Ca2+ ATPase with this method had 9 transmembrane co-translational insertion domains and coupled with other evidence these data resulted in a 11 transmembrane segment model. Translation of segments of the g astric H+,K+ ATPase provided evidence for only 7 transmembrane segment s but coupled with other data established a 10 membrane segment model The G7 protein, the CCK-A receptor showed the presence of 6 of the 7 t ransmembrane segments postulated for this protein. Translation of segm ents of the human ileal bile acid transporter showed the presence of 8 membrane insertion domains. However, translation of the intact protei n provided evidence for an odd number of transmembrane segments, resul ting in a tentative model containing 7 or 9 transmembrane segments. Ne ither G7 type protein appeared to have an arrangement of sequential to pogenic signals consistent with the final assembled protein. This meth od provides a useful addition to methods of determining membrane domai ns of integral membrane proteins but must in general be utilized with other methods to establish the number of transmembrane alpha-helices.