COMPARATIVE TOPOLOGY STUDIES IN SACCHAROMYCES-CEREVISIAE AND IN ESCHERICHIA-COLI - THE N-TERMINAL HALF OF THE YEAST ABC PROTEIN STE6

Gene fusions have provided a strategy for determining the topology of polytopic membrane proteins in Escherichia coli. To valuate whether th is highly effective approach is applicable to heterologously expressed eukaryotic integral membrane proteins, we have carried out a comparat ive topological study of the eukaryotic membrane protein Ste6 both in bacteria and in yeast, Ste6, is an ATP binding cassette (ABC) protein essential for export of the a-factor mating pheromone in Saccharomyces cerevisiae, The topogenic reporters, invertase in S. cerevisiae and a lkaline phosphatase in E. coli, were fused to Ste6 at identical sites and the fusions were expressed in yeast and bacteria, respectively. Th e results obtained in both systems are similar, although more definiti ve in E. coli, and support the predicted six-transmembrane spans organ ization of the N-terminal half of Ste6, Thus, the topological determin ants for membrane insertion of polytopic proteins in prokaryotic and i n eukaryotic systems appear to be highly similar, In this study we als o demonstrate that Ste6 does not contain a cleaved signal sequence.