The use of gene-fusions to determine membrane protein topology in Saccharomyces cerevisiae.

We have used protein-fusions to study in Saccharomyces cerevisiae the topology and integration of arginine permease. Since this membrane protein does not contain a cleavable signal sequence, we sought to identify the first internal signal by, initially, fusing the cytoplasmic enzyme, galactokinase, to various positions along the amino-terminal region, and then measuring in vitro the translocation of galactokinase across the membrane of the endoplasmic reticulum. Five fusion proteins were examined that contained, progressively, zero to four hydrophobic segments. The galactokinase moiety of fusion 5, but not fusions 1-4, was translocated. Fusion 4 differed from 5 by only the fourth hydrophobic segment, indicated that this region contains the first internal signal. From this we conclude that hydrophobic segment IV spans the membrane, and that the hydrophilic domain amino-terminal to it lies on the cytoplasmic side of the membrane. If any of the first three segments actually span the membrane, then they probably integrate by a mechanism that differs from segment IV.