Control of cellular trafficking of mammalian voltage sensitive phosphatase (VSP) through protein-protein interactions

Voltage-sensitive phosphatases (VSPs) possess a unique combination of a voltage sensor domain (VSD), akin to those found in voltage-gated ion channels (VGICs), and a catalytic domain similar to the tumor suppressor protein PTEN. This fusion of voltage sensing ability and lipid phosphatase activity makes them exceptional signaling enzymes that convert phosphoinositide’s (PIs) in response to depolarization of the membrane potential. Thus, VSPs are known to directly link cellular electrical signaling to intracellular pathways. VSPs exist in several species. However, the physiological role of VSPs remains largely unknown, except for a role in regulating sperm motility in mammals. The remarkable voltage dependent phosphatase activity against PIs has been characterized functionally for VSPs from few non-mammalian species e.g., Ciona intestinalis (Ci-VSP), facilitated by robust expression and plasma membrane (PM) targeting in heterologous expression systems. In contrast, mammalian VSPs are poorly understood due to their inability to reach the PM upon over-expression. This has led to speculation about their ability to dephosphorylate PIs upon membrane depolarization. The lack of reliable antibodies complicates the understanding of the physiological role of VSPs in the native environment. In my PhD thesis, we aimed to perform an unbiased screening approach to uncover mechanisms of PM targeting of the VSP from mouse (mVSP) which in turn enabled examination of its electrochemical function in an over-expression system. Using a membrane-based yeast two-hybrid screening, we identified the single-transmembrane-domain protein Basigin (BSG) as a putative protein-protein interaction partner of mVSP. Through co-expression experiments in a heterologous system, we found that BSG acts as an ancillary protein subunit for mVSP, which mediates trafficking from the endoplasmic reticulum (ER) to the PM. PM targeting of mVSP was also observed with other BSG family members, namely Neuroplastin (NPTN) and Embigin (EMB). In order to gain a deeper understanding of the precise regions or domains of BSG that are involved in the PM targeting of mVSP, we took a mutational approach. Our investigations revealed that the single transmembrane region of BSG alone is sufficient to facilitate the trafficking of mVSP to the PM. Vice versa, we explored the regions of mVSP that determine intracellular localization in the absence of BSG. By employing a chimeric approach, we successfully identified that both the N and C-terminal sequence stretches of mVSP are responsible for its retention within the intracellular compartment. Through the facilitation of BSG-mediated membrane trafficking, we were able to uncover the function of mVSP, which demonstrated its function as a voltage-activated lipid phosphatase. Specifically, mVSP is a 5'-phosphatase of PI(4,5)P2 and PI(3,4,5)P3 activated by depolarization of the membrane potential. Notably, mVSP and BSG were previously shown to be localized in the sperm tail and have been implicated in the regulation of sperm motility. These findings strongly suggest that BSG plays a critical role in targeting mVSPs within the sperm tail, thereby influencing sperm motility by modulating PI metabolism.