A basic requirement of all cells is the ability to sense and respond to changes in the environment. Osmolarity is one of the most basic conditions to which cells must respond. Despite the ubiquitous nature of osmosensing systems, the molecular mechanism by which osmotic pressure is sensed is largely unknown. Studies involving microbial model systems have played an important role in identifying the sensors and signal transduction pathways that respond to changes in osmolarity. We plan to use the yeast osmotic stress sensor, Slnlp, as a paradigm for eukaryotic osmosensors. Changes in osmotic conditions regulate Slnlp causing changes in phosphate flux between the individual proteins comprising the two-component signaling system. The fundamental question addressed by this proposal is how Slnlp activity is regulated in response to changes in osmolarity. Using computational, genetic, and biochemical techniques, we have identified a coiled-coil dimerization domain that plays a key role in mediating the stimulus-activation step. It is located in the linker region between the membrane and the kinase domain. The specific objective of this application is to perform a detailed structure-function analysis of the coiled coil (CC) region of the yeast Slnlp osmosensor to test the hypothesis that the unusual composition of the HK CC contributes to the regulation of the HK family of sensor kinases.
The specific aims of the proposal are to (1) Genetically dissect the Sin1 CC domain, (2) Determine the structure of the CC domain and CC mutants, and (3) Develop membrane-based assays for Sin1 function. The elucidation of the unique structural and mechanistic features of the two-component type coiled-coil domain in Slnlp will serve as a model for this class of signaling molecules and may lead to the development of histidine kinase inhibitors for antifungal therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM068746-03
Application #
7011922
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Anderson, James J
Project Start
2003-08-01
Project End
2007-07-31
Budget Start
2005-01-01
Budget End
2005-07-31
Support Year
3
Fiscal Year
2004
Total Cost
$123,900
Indirect Cost
Name
Medical College of Wisconsin
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
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