A ci-VSP/Tpip Chimera Exhibiting Voltage-Dependent Pi(4,5)P2/PIP3-5’-Phosphatase Activity

Abstract

Phosphoinositide (PI) concentrations in cell membranes play an important role in many cellular processes. PI phosphatases are crucial for the regulation of these concentrations. Among these phosphatases are the voltage-sensitive phosphatases (VSPs) like the prototypical Ci-VSP, which provide a new paradigm for the control of enzymatic activity. Recently, we showed that voltage sensitivity can be conferred to the cytoplasmic phosphatase PTEN: by fusing the voltage sensor domain (VSD) of Ci-VSP to the catalytic domain (CD) of PTEN, we generated an engineered chimeric VSP, Ci-VSPTEN. Here we extend this work and report on a chimera created by fusing Ci-VSP's VSD to the CD of the putative PI-phosphatase TPIPα. We demonstrate that this Ci-VSP/TPIP chimera exhibits voltage dependent enzymatic activity. The substrate and position specificity of Ci-VSP/TPIP is analyzed using genetically encoded PI-specific fluorescence labeled probes, the membrane binding of which is assessed using total internal reflection fluorescence (TIRF) microscopy. Control over the membrane voltage is achieved by whole-cell patch clamping. We find that upon depolarization, the membrane binding of the PIP3 specific probe, Btk-PH, and the PI(4,5)P2 specific probe, PLCδ1-PH, decreases, when co-expressed with Ci-VSP/TPIP. In contrast, membrane binding of the PI(3,4)P2 specific TAPP1-PH and the PI(4)P specific OSBP-PH probes increases upon depolarization. These findings identify the Ci-VSP/TPIP chimera as a voltage-sensitive PI(4,5)P2/PIP3-5’-phosphatase. We conclude that TPIPα is a 5’-phosphatase in-vivo, in contrast to PIP3-3’-phosphatase activity in-vitro reported previously (Walker et al., Biochem. J. (2001) 360, 277 −283). These data will help to further the understanding of the mechanism by which voltage control is exerted in VSPs. Additionally, our experiments demonstrate the usefulness of engineered VSPs as a novel tool for the analysis of PI-phosphatases in-vivo, an obligatory complement to in-vitro characterization. Supported by Deutsche Forschungsgemeinschaft (SFB593 TPA12 to D.O.)