In Vivo Topological Analysis of Ste2, a Yeast Plasma Membrane Protein, by Using β-Lactamase Gene Fusions

Abstract

Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae α-factor, and βla, the secreted form of β-lactamase encoded by the bla gene of pBR322. The Ste2 and βla components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in βla secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of βla; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in βla secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of βla activity occurred, which is consistent with inversion of the orientation of the βla reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced βla secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of βla detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.