Date of Award

5-2013

Document Type

Campus Access Thesis

College

College of Theology, Arts, & Sciences

Department

Math & Science

Degree Name

Biology, BA

First Supervisor

Mihail Iordanov, Ph.D.

Keywords

scaffold proteins

Abstract

Cells use scaffold proteins to bind and organize components of signaling pathways. A customized signaling response underlies biological adaptation. Changes in scaffold protein genes may be a mechanism through which organisms customize signaling response through evolution. Such a hypothesis has emerged: scaffold proteins have served as evolutionary test beds for organisms to elaborate on existing pathways to generate customized signaling mechanisms. However, direct evidence for the evolutionary diversification of scaffolds is lacking. Ste5 is a scaffold in the mating pathway of S. cerevisiae with orthologues in related species. Ste5 binds and organizes a kinase cascade, where phosphorylation of the terminal kinase (Fus3) by its upstream kinase (Ste7) activates the mating response. However, the Fus3-binding domain (FBD) on Ste5 binds Fus3 to stimulate autophosphorylation and this phospho-Fus3 inhibits pathway activation. This regulatory mechanism may be a recently evolved feature of the scaffold, since little is known about FBD or Fus3 orthologues. Therefore, we hypothesized that selected FBD orthologues will not stimulate phosphorylation of S. cerevisiae Fus3 and that this would affect pathway output in vivo. Using an in vitro system, we found that all FBD orthologues failed to stimulate Fus3 phosphorylation, except one V. polyspora, a related yeast. Further investigation revealed that stimulation of phosphorylation by the V. polyspora FBD is kinetically distinct from the S. cerevisiae FBD. To test for consequences in vivo, chimeric Ste5 proteins were expressed in yeast and fluorescence-activated cell sorting (FACS) was used to monitor pathway activity. However, variability in Ste5 expression levels confounded interpretation of the FACS data. Though direct pathway output could not be quantified, our data point to an evolutionary expansion of Ste5 function in S. cerevisiae and imply that scaffold evolution can underlie customized signaling responses.

Honors: Thesis with Distinction Award

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