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MBC in Press, published online ahead of print January 12, 2005
Mol. Biol. Cell 10.1091/mbc.E04-07-0642

A more recent version of this article appeared on March 1, 2005
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Submitted on July 28, 2004
Revised on December 10, 2004
Accepted on January 1, 2005

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

Holly D. Johnston, Christopher Foote, Andrea Santeford,* and Steven F. Nothwehr{dagger}

Division of Biological Sciences, University of Missouri, Columbia, MO 65211

Monitoring Editor: Benjamin Glick

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-alkaline phosphatase, 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-alkaline phosphatase to Ala was found to block trafficking to the prevacuolar compartment (PVC) while 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 ER-to-Golgi transport of newly synthesized A(S13A)-alkaline phosphatase was indistinguishable from wild-type indicating that the lack of transport of A(S13A)-alkaline phosphatase to the PVC was instead due to differences in Golgi/endosomal trafficking. The A(S13A)-alkaline phosphatase protein exhibited a TGN-like localization similar to that of wild-type A-alkaline phosphatase. 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-alkaline phosphatase and imply that phosphorylation of S13 may regulate recognition of A-alkaline phosphatase by vesicular trafficking machinery.

*Present address: Dept. of Biology, St. Louis University, Macelwane Hall, 3507 Laclede Ave., St. Louis, MO 63103-2010.

{dagger}Corresponding author. E-mail: nothwehrs{at}missouri.edu






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