Golgi-to-late endosome trafficking of the yeast pheromone processing enzyme Ste13p is regulated by a phosphorylation site in its cytosolic domain

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 S-13 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 S-13 residue was shown by mass spectrometry to be phosphorylated. The rate of endoplasmic reticulum-to-Golgi transport of newly synthesized A(S(13)A)-ALP was indistinguishable from wild-type, indicating that the lack of transport of A(S(13)A)-ALP to the PVC was instead due to differences in Golgi/endosomal trafficking. The A(S(13)A)-ALP protein exhibited a TGN-like localization similar to that of wild-type A-ALP. Similarly, the S(13)A mutation in endogenous Ste13p did not reduce the extent of or longevity of its localization to the TGN as shown by a-factor processing assays. These results indicate that S-13 phosphorylation is required for TGN-to-PVC trafficking of A-ALP and imply that phosphorylation of S-13 may regulate recognition of A-ALP by vesicular trafficking machinery.