The Role of the C2 Domain of Voltage Sensing Phosphatase (VSP)

Abstract

The voltage-sensing phosphatase (VSP) is a voltage-activated enzyme that dephosphorylates phosphatidylinositol phosphates (PIPs). VSP is unique because it is the only example of an enzyme activated by voltage, providing the first direct link between lipid signaling pathways and membrane potential. Along with the voltage sensing domain, VSP also has a catalytic domain and a C2 domain. To date, the role of the VSP C2 domain is not understood. C2 domains are generally known as lipid binding domains, however, previous work has suggested the VSP C2 has a role in catalytic activity. Specifically, the crystal structures [1,2] show the 522-loop of the C2 forms a portion of the enzyme active site and the mutation Y522A altered enzyme activity [2]. We further probed the role of Y522 by introducing a phenylalanine instead of an alanine. We found the Y522F mutation also shifts the voltage dependence of catalytic activity, suggesting that hydrogen bonding is not a factor when this residue participates in VSP activity. A role in catalysis does not exclude the VSP C2 from also contributing to lipid binding. To investigate this possibility, we started by fully deleting the C2 domain from Ci-VSP and found that the presence of the full C2 domain is necessary for normal phosphatase function. We then combined more specific C2 mutations with the ability to manipulate PIP concentrations using a rapamycin-induced system in order to further address the possible roles of the C2 in VSP function. Our goal is to investigate how the C2 domain contributes to VSP function.[1] Matsuda, M. et al. JBC, 286, 23368-77 (2011).[2] Liu, L. et al. NSMB, 19, 633-641 (2012).