Mutational analysis of the role of N-glycosylation in alpha-factor receptor function

The alpha -factor mating pheromone receptor (encoded by STE2) activates a G protein signaling pathway that stimulates the conjugation of Saccharomyces cerevisiae yeast cells. The alpha -factor receptor is known to undergo sev eral forms of post-translational modification, including phosphorylation, m ono-ubiquitination, and N-linked glycosylation. Since phosphorylation and m ono-ubiquitination have been shown previously to play key roles in regulati ng the signaling activity and membrane trafficking of the alpha -factor rec eptors, the role of N-linked glycosylation was investigated in this study. The Asn residues in the five consensus sites for N-linked glycosylation pre sent in the extracellular regions of the receptor protein were mutated to p revent carbohydrate attachment at these sites. Mutation of two sites near t he receptor N-terminus (N25Q and N32Q) diminished the degree of receptor gl ycosylation, and the corresponding double mutant was not detectably N-glyco sylated. The nonglycosylated receptors displayed normal function and subcel lular localization, indicating that glycosylation is not important for wild -type receptor activity. However, mutation of the glycosylation sites resul ted in improved plasma membrane localization for the Ste2-3 mutant receptor s that are normally retained intracellularly at elevated temperatures. Thes e results suggest that N-glycosylation may be involved in the sorting proce ss for misfolded Ste2 proteins, and may similarly affect certain mutant rec eptors whose altered trafficking is implicated in human diseases.