A method for determining the in vivo topology of yeast polytopic membrane proteins demonstrates that Gap1p fully integrates into the membrane independently of Shr3p

The general amino acid permease (Gap1p) of Saccharomyces cerevisiae is an i ntegral membrane protein that contains 12 hydrophobic regions predicted to be membrane-spanning segments. A topological reporter construct, encoding a n internal 53-amino acid peptide of invertase (Suc2p) containing three Asp- X-Ser/Thr glycosylation sites, was inserted in-frame into the hydrophilic N H2- and COOH-terminal domains and each of the 11 hydrophilic loops that sep arate the 12 hydrophobic segments of Gap1p. The resulting 13 gene sandwich fusion proteins were expressed in a gap1 Delta null mutant strain; 9 of the se retain amino acid transport activity and are folded and correctly target ed to the plasma membrane. The glycosylation state of each of the fusion pr oteins was monitored; the results indicate that all 12 hydrophobic segments of Gap1p span the membrane, and the MI, and COOH termini are cytoplasmical ly oriented. These results were independently tested by isolating sealed ri ght-side out microsomes from sec12-1 strains expressing six different Gap1p constructs containing functional factor Xa protease cleavage sites. The pa ttern of factor Xa protease cleavage was found to be consistent with the pr esence of 12 membrane-spanning domains. Gap1p exhibited the same membrane t opology in strains lacking Shr3p; therefore, Gap1p fully integrates into th e ER membrane independently of this permease-specific packaging chaperone.