Golgi-to-Late Endosome Trafficking of the Yeast Pheromone Processing Enzyme Ste13p Is Regulated by a Phosphorylation Site in its Cytosolic Domain

摘要

This study addressed whether phosphorylation regulates trafficking of yeast membrane proteins that cycle between the trans -Golgi network (TGN) and endosomal system. The TGN membrane proteins A-ALP, a model protein containing the Ste13p cytosolic domain fused to alkaline phosphatase (ALP), and Kex2p were found to be phosphorylated in vivo. Mutation of the S₁₃ residue on the cytosolic domain of A-ALP to Ala was found to block trafficking to the prevacuolar compartment (PVC), whereas a S₁₃D mutation generated to mimic phosphorylation accelerated trafficking into the PVC. The S₁₃ residue was shown by mass spectrometry to be phosphorylated. The rate of endoplasmic reticulum-to-Golgi transport of newly synthesized A(S₁₃A)-ALP was indistinguishable from wild-type, indicating that the lack of transport of A(S₁₃A)-ALP to the PVC was instead due to differences in Golgi/endosomal trafficking. The A(S₁₃A)-ALP protein exhibited a TGN-like localization similar to that of wild-type A-ALP. Similarly, the S₁₃A mutation in endogenous Ste13p did not reduce the extent of or longevity of its localization to the TGN as shown by α-factor processing assays. These results indicate that S₁₃ phosphorylation is required for TGN-to-PVC trafficking of A-ALP and imply that phosphorylation of S₁₃ may regulate recognition of A-ALP by vesicular trafficking machinery.