Identifying novel roles for Fus3p in cell fusion and mating.
Dina Matheos (1), Kevan Shokat (2), Eric Phizicky (3), Mark
Rose (4)
(1) Department of Molecular Biology, Princeton University, Princeton, NJ
08544;
(2) Department of Cellular & Molecular Pharmacology, UCSF, San
Francisco, CA 94143;
(3) Department of Biochemistry & Biophysics, Univeristy of Rochester
School of Medicine, Rochester, NY 14642;
(4) Molecular Biology, Princeton University, Washington Rd, Princeton,
NJ 08544, USA
In S. cerevisiae, conjugation is initiated by an intracellular
signal transduction cascade that is required for morphological changes,
G1 arrest, and transcriptional activation. The MAP kinase Fus3p has well
characterized roles in transcriptional activation and G1 arrest, but the
null mutant also implicates Fus3p in the process of cell fusion. Because
Fus3p is a kinase, we have chosen to address its role in cell fusion by
identifying Fus3p substrates. First, I have performed a two-hybrid
screen using a kinase dead allele of Fus3p. Substrate-kinase
interactions are transient, but a catalytically crippled kinase may
interact with a substrate more stably, resulting in facilitated target
detection. I identified Kre6p as a Fus3p interacting protein suggesting
that Fus3p may be required to regulate beta-glucan synthesis during cell
fusion. As a secondary approach, I am using an in vitro kinase
assay to look directly at Fus3p substrates. I have assayed proteins that
have known functions in mating or that may logically be involved as
accessories in mating. This approach has yielded Bni1p, Rho4p and Rho5p
as in vitro substrates, uncovering a function for Fus3p in
polarity during mating. Through screening a GST fusion library, I
have identified Hif1p and Ypl247p as substrates. These substrates have
begun to delineate a Fus3p cell fusion pathway and potentially novel
functions for Fus3p.
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