The membrane topology of the rat endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) Ca 2؉ATPases were investigated using in vitro transcription/ translation of fusion vectors containing DNA sequences encoding putative membrane-spanning domains. The sequences of these Ca A construct containing the fifth predicted transmembrane segment was able to act only as a stop transfer sequence. The sixth transmembrane segment did not insert cotranslationally into the membrane. The seventh was able to act as both a signal anchor and stop transfer sequence, and the eighth showed stop transfer ability in the M1 vector. The ninth transmembrane segment had both signal anchor and stop transfer capacity, whereas the tenth transmembrane segment showed only stop transfer sequence properties. The eleventh transmembrane sequence, unique to the ER Ca 2؉ ATPase, had both signal anchor and stop transfer properties. These translation data provide direct experimental evidence for 8 or 9 of the 10 or 11 predicted transmembrane sequences in the current topological models for the SR or ER Ca 2؉ ATPases, respectively.

No abstract

The ␣ subunit of eukaryotic P-type ATPases has ten experimentally defined transmembrane or membrane inserted segments. The fifth and sixth of these are short, not predicted by hydropathy analysis, do not insert independently into microsomal membranes, and are readily removed after tryptic digestion and therefore may be membrane inserted sequences. Acid transport by the gastric H,K-ATPase is covalently inhibited by several substituted pyridyl methylsulfinyl benzimidazoles, such as omeprazole. These act as probes of accessible extracytoplasmic thiols because they are accumulated in the acid transporting gastric vesicles and then convert to thiol reactive, cationic tetracyclic sulfenamides. Inhibition is due mainly to disulfide formation with

The transmembrane topology of Na(+)/H(+) exchanger NHE3 has been studied using in vitro transcription/translation of two types of fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NHE3 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 region and a reporter sequence containing five N-linked glycosylation consensus 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 putative msd in both the HKM0 and the HKM1 vectors. The linker region of the vectors was replaced by sequences that contain putative msds of NHE3 individually or in pairs. In vitro transcription/translation was performed using [(35)S]methionine in a reticulocyte lysate system +/- microsomes, and the translation products were identified by autoradiography following separation using SDS-PAGE. We propose a revised NHE3 topology model, which contains a cleaved signal peptide followed by 11 msds, including extracellular orientation of the N-terminus and intracellular orientation of the C-terminus. The presence of a cleavable signal peptide in NHE3 was demonstrated by its cleavage from NHE3 during translational processing of full-length and truncated NHE3 in the presence of microsomes. Of 11 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 alone. Of the latter, 3, 5, 6, and 9 were shown to act as stop transfer sequences after C-terminal extension. The actual membrane orientation of each sequential transmembrane segment of NHE3 was deduced from the membrane location 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 extracellular loops, did not act as msds when tested alone. However, the extension of the fifth extracellular loop with adjacent putative msds showed some membrane-associated properties suggesting that the fifth extracellular loop might be acting as a "P-loop"-like structure.