N-terminal chimeric constructs improve the expression of sarcoplasmic reticulum Ca2+-ATPase in yeast

AbstractWild-type and chimeric constructs comprising rabbit sarcoplasmic reticulum (SR) Ca2+-ATPase and the N-terminal cytoplasmic portion of yeast plasma membrane H+-ATPase were expressed in yeast under control of a heat-shock regulated promoter. The wild-type ATPase was found predominantly in endoplasmic reticulum (ER) membranes. Addition of the first 88 residues of H+-ATPase to the Ca2+-ATPase N-terminal end promoted a marked shift in the localization of chimeric H+/Ca2+-ATPase which accumulated in a light membrane fraction associated with yeast smooth ER. Furthermore, there was a three-fold increase in the overall level of expression of chimeric H+/Ca2+-ATPase. Similar results were obtained for a chimeric Ca2+-ATPase containing a hexahistidine sequence added to its N-terminal end. Both H+/Ca2+-ATPase and 6×His-Ca2+-ATPase were functional as demonstrated by their ability to form a phosphorylated intermediate and undergo fast turnover. Conversely, a replacement chimera in which the N-terminal end of SR Ca2+-ATPase was replaced by the corresponding segment of H+-ATPase was not stably expressed in yeast membranes. These results indicate that the N-terminal segment of Ca2+-ATPase plays an important role in enzyme assembly and contains structural determinants necessary for ER retention of the ATPase