Introductory Lecture: In Vitro Translation Analysis of Integral Membrane Proteins

Abstract

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⁺ ATPase, the CCK-A receptor and the human ileal bile acid transporter. This method 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 β-subunit of the gastric H⁺,K⁺ ATPase. The latter contains 5 potential N-linked glycosylation sites. Translation of vectors containing the cDNA encoding one, two or more putative transmembrane domains in the absence or presence of microsomes allowed determination of signal anchor or stop transfer properties of the putative transmembrane domains by the molecular weight shift on SDS PAGE. The P type ATPase from Helicobacter pylori showed the presence of 8 transmembrane segments with this method. The SERCA 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 gastric H⁺,K⁺ ATPase provided evidence for only 7 transmembrane segments 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 transmembrane segments postulated for this protein. Translation of segments of the human ileal bile acid transporter showed the presence of 8 membrane insertion domains. However, translation of the intact protein provided evidence for an odd number of transmembrane segments, resulting in a tentative model containing 7 or 9 transmembrane segments. Neither G7 type protein appeared to have an arrangement of sequential topogenic signals consistent with the final assembled protein. This method provides a useful addition to methods of determining membrane domains of integral membrane proteins but must in general be utilized with other methods to establish the number of transmembrane α-helices.