In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.
Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la 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 beta la 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 beta la; 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 beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la 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 beta la 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.
Cartwright
C P
CP
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical Center, Worcester 01655.
Tipper
D J
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics*; D008961:Models, Structural; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics*
D005656:Fungal Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases