Na+/H+ exchanger NHE3 has 11 membrane spanning domains and a cleaved signal peptide: Topology analysis using in vitro transcription/translation

The transmembrane topology of Na+/H+ exchanger NHE3 has been studied using in vitro tfanscription/translation of two types of fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative N HE3 membrane spanning domains (msds), These vectors encode N-terminal 101 ( HKM0) or 139 (HKM1) amino acids of the H,K-ATPase alpha-subunit, a linker r egion and a reporter sequence containing five N-linked glycosylation consen sus sites in the C-terminal 177 amino acids of the H,K-ATPase beta-subunit. The glycosylation status of the reporter sequence was used as a marker for the analysis of signal anchor and stop transfer properties of each putativ e msd in both the HKM0 and the HKM1 vectors. The linker region of the vecto rs was replaced by sequences that contain putative msds of NHE3 individuall y or in pairs. In vitro transcription/translation was performed using [S-35 ]methionine in a reticulocyte lysate system +/- microsomes, and the transla tion products were identified by autoradiography following separation using SDS-PAGE, We propose a revised NHE3 topology model, which contains a cleav ed signal peptide followed by 11 msds, including extracellular orientation of the N-terminus and intracellular orientation of the C-terminus. The pres ence of a cleavable signal peptide in NHE3 was demonstrated by its cleavage from NHE3 during translational processing of full-length and truncated NHE 3 in the presence of microsomes. Of ii putative msds, six (msds 1, 2, 4, 7, 10, and 11) acted as both signal anchor and stop transfer sequences, while five (msds 3, 5, 6, 8, and 9) had signal anchor activities when tested alo ne. Of the latter, 3, 5, 6, and 9 were shown to act as stop transfer sequen ces after C-terminal extension. The actual membrane orientation of each seq uential transmembrane segment of NHE3 was deduced from the membrane locatio n of the N- and C-termini of NHE3. The regions between putative msds 8 and 9 and between msds 10 and 11, which correspond to the fourth and fifth extr acellular loops, did not act as msds when tested alone. However, the extens ion of the fifth extracellular loop with adjacent putative msds showed some membrane-associated properties suggesting that the fifth extracellular loo p might be acting as a "P-loop"-like structure.