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 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 alpha-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.
Johnston
Holly D
HD
Division of Biological Sciences, University of Missouri, Columbia, MO 65211.
Foote
Christopher
C
Santeford
Andrea
A
Nothwehr
Steven F
SF
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010675:Pheromones / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D043484:Proprotein Convertases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D013347:Subcellular Fractions; D013997:Time Factors; D021601:trans-Golgi Network / Q000378:metabolism
D010675:Pheromones; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000469:Alkaline Phosphatase; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C061833:STE13 protein, S cerevisiae; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae