C-terminal sequences can inhibit the insertion of membrane proteins into the endoplasmic reticulum of Saccharomyces cerevisiae

Abstract

We have constructed three gene fusions that encode portions of a membrane protein, arginine permease, fused to a reporter domain, the cytoplasmic enzyme histidinol dehydrogenase (HD), located at the C-terminal end. These fusion proteins contain at least one of the internal signal sequences of arginine permease. When the fusion proteins were expressed in Saccharomyces cerevisiae and inserted into the endoplasmic reticulum (ER), two of the fusion proteins placed HD on the luminal side of the ER membrane, but only when a piece of DNA encoding a spacer protein segment was inserted into the fusion joint. The third fusion protein, with or without the spacer included, placed HD on the cytoplasmic side of the membrane. These results suggest that (i) sequences C-terminal to the internal signal sequence can inhibit membrane insertion and (ii) HD requires a preceding spacer segment to be translocated across the ER membrane.