Abstract
The ability of a cell to rapidly remodel its actin cytoskeleton is critical for many of its cellular functions. Actin exists in monomeric and filamentous fonns and cycles between these fonns in a process known as actin turnover. A TP-bound actin monomers polymerize into a filament, and as the filament ages, become ADP bound. When actin filaments are dcpolymerizcd by ADF/cofilin, which binds actin monomers and inhibits nucleotide exchange, apool of cotllinbound ADP-actin monomers is rapidly produced. Therefore, cells require additional factors to promote actin recycling, converting ADP-actin back to assembly-competent ATP-actin. Previous· work in our lab showed that a critical player in recycling is Srv2/CAP; however, its mechanism is still not understood. Srv2/CAP has four separate functional domains, each with different binding partners and proposed functions. However, the functional relationship between these domains in the process of actin recycling is unclear. In my thesis work, I performed a genetic and biochemical dissection of Srv2/CAP and unexpectedly found that the two halves of the protein can function efficiently in trans to promote recycling. However, when I bisected the protein at a different boundary, the two halves were no longer functional. These biochemical results were mirrored in vivo when 1 tested the ability of the fragments to complement SRV2 function in yeast. These data suggest that specific domains of Srv2/CAP must be physically linked to function, while others do not, which important implications for Srv2/CAP mechanism.