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 several forms of post-translational modification, including phosphorylation, mono-ubiquitination, and N-linked glycosylation. Since phosphorylation and mono-ubiquitination have been shown previously to play key roles in regulating the signaling activity and membrane trafficking of the alpha-factor receptors, the role of N-linked glycosylation was investigated in this study. The Asn residues in the five consensus sites for N-linked glycosylation present in the extracellular regions of the receptor protein were mutated to prevent carbohydrate attachment at these sites. Mutation of two sites near the receptor N-terminus (N25Q and N32Q) diminished the degree of receptor glycosylation, and the corresponding double mutant was not detectably N-glycosylated. The nonglycosylated receptors displayed normal function and subcellular localization, indicating that glycosylation is not important for wild-type receptor activity. However, mutation of the glycosylation sites resulted in improved plasma membrane localization for the Ste2-3 mutant receptors that are normally retained intracellularly at elevated temperatures. These results suggest that N-glycosylation may be involved in the sorting process for misfolded Ste2 proteins, and may similarly affect certain mutant receptors whose altered trafficking is implicated in human diseases.
Mentesana
P E
PE
Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook 11794-5222, USA.
Konopka
J B
JB
D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D017930:Genes, Reporter; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D014157:Transcription Factors
D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors