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 evaluate whether this highly effective approach is applicable to heterologously expressed eukaryotic integral membrane proteins, we have carried out a comparative 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 alkaline phosphatase in E. coli, were fused to Ste6 at identical sites and the fusions were expressed in yeast and bacteria, respectively. The results obtained in both systems are similar, although more definitive in E. coli, and support the predicted six-transmembrane spans organization of the N-terminal half of Ste6. Thus, the topological determinants for membrane insertion of polytopic proteins in prokaryotic and in eukaryotic systems appear to be highly similar. In this study we also demonstrate that Ste6 does not contain a cleaved signal sequence.
Geller
D
D
Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Taglicht
D
D
Edgar
R
R
Tam
A
A
Pines
O
O
Michaelis
S
S
Bibi
E
E
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D000469:Alkaline Phosphatase / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D005798:Genes, Bacterial; D005800:Genes, Fungal; D017930:Genes, Reporter; D006023:Glycoproteins; D006026:Glycoside Hydrolases / Q000235:genetics; D006031:Glycosylation; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D043324:beta-Fructofuranosidase
D018528:ATP-Binding Cassette Transporters; D005656:Fungal Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000469:Alkaline Phosphatase; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase