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A part of the Alpha Project, I worked at the Molecular Sciences
Institute in collaboration with Pacific Northwest National
Laboratory to identify sites of phosphorylation on proteins
in the S. cerevisiae mating pheromone pathway by mass
spectrometry. Using the TAP-tagged yeast collection,
we purified 14 of the key signal transduction proteins individually
from both pheromone-treated and untreated cells. Next, we performed
a series of experiments where we mixed all the strains together,
added pheromone, collected time points and purified all 14
proteins at once for the purpose of relative quantification
and timing of events. We were able to identify over 100 sites
of phosphorylation across the different pathway proteins in
response to pheromone and characterize the phosphorylation
dynamics in the pathway. We verified the biological relevance
of several of the sites of phosphorylation by site-directed
mutagenesis followed by quantitative pheromone response assays.
This is the first single study to exhaustively catalog phosphorylation-dependent
functional connections through an entire signaling pathway.
Ghaemmaghami, S., Huh, W. K., Bower, K., Howson, R. W., Belle,
A., Dephoure, N., O'Shea, E. K., and Weissman, J. S. (2003).
Global analysis of protein expression in yeast. Nature 425,
737-41.
Maxwell, R., Resnekov, O., Pincus, D., Benjamin, K., Smith,
D., and Brent, R. Phosphorylation-dependent functional connections
among proteins in an information processing pathway. In preparation.
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