中国生物工程杂志

Members of the G protein-coupled receptor (GPCR) family play important roles in the sensing of extracellular signals. Ste2 is one of the three GPCR proteins in the budding yeast, Saccharomyces cerevisiae. In the past years, extensive efforts have focused on how the function and expression of Ste2 are affected by various mutations. However, little is known about the mechanisms dictating its proper subcellular localization. To this end, a series of mutants containing deletions or substitutions in the N-terminus, C-terminus, transmembrane domains, intra/extracellular loops are constructed. The subcellular localization of wild-type and mutant Ste2 proteins are observed by fluorescent microscopy, in combination with a set of organelle markers, to determine their localization. Wild-type Ste2 is primarily targeted to the plasma membrane and vacuolar lumen. Deletion of the C-terminus eliminates the vascular signal, targeting the protein to the plasma membrane and endoplasmic reticulum instead. The result is similar when the C-terminus is substituted by the corresponding regions from ORI7 and OR17-40, two mammalian GPCRs. When the N-terminus, first extracellular loop (EL1), the second extracellular loop (EL2) or the third intracellular loop (IL3) is substituted, plasma membrane targeting of Ste2 is substantially attenuated or eliminated. Some of these mutants accumulate on intracellular punctate structures. These results suggest that the N-terminus, EL1, EL2 and IL3 regions contain potential sorting signals regulating the transport of Ste2 to the plasma membrane, and that the C-terminus contains signals for its targeting to the vacuole. The present work provides new insights towards understanding the mechanisms governing GPCR protein subcellular localization.