N-terminal domain of Gpa1 (G protein alpha subunit) is sufficient for plasma membrane targeting in yeast Saccharomyces cerevisiae

G proteins play a central role in transmitting signals from cell surface re ceptors to effector proteins inside the cell. Signaling can only occur, how ever, if all these protein components are properly assembled and localized at the plasma membrane. Past studies have shown that certain segments withi n the N-terminal region of the G protein a subunit are necessary for membra ne attachment. Here we identify a region within the yeast G alpha (Gpa1) th at is sufficient for membrane attachment, as well as for specific targeting to the plasma membrane. Initially, we constructed chimeric proteins that r eplace the N terminus of mammalian Gsa with the corresponding sequence from Gpa1, Gsa is inefficiently targeted to the yeast plasma membrane and there fore cannot fully complement the loss of Gpa1, Gpa1-G(s)alpha chimeras were assayed for proper membrane localization by functional complementation of a gpa1 Delta mutant, and by sucrose density gradient fractionation of cell membranes. Most of the chimeras tested, including one with only the N-termi nal 7 amino acids from Gpa1, exhibited normal membrane targeting and comple menting activity. We also fused various lengths of N-terminal Gpa1 sequence to glutathione-S-transferase (GST), a heterologous protein normally expres sed in the cytoplasm, The first 67- 36- or 9-amino acids of Gpa1 were all s ufficient to direct GST specifically to the plasma membrane in yeast. This analysis defines the extreme N terminus of Gpa1 as the primary determinant of proper membrane targeting, and represents an essential step towards isol ating and identifying G protein-targeting proteins within the plasma membra ne.