Homeostatic regulation of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2) in receptor-stimulated cells is mediated by the lipid transfer protein Nir2. Nir2 is dynamically recruited to endoplasmic reticulum-plasma membrane (ER-PM) junctions to facilitate replenishment of PM PIP2 hydrolyzed during receptor-mediated signaling. However, our knowledge regarding the activation and sustainment of Nir2-mediated replenishment of PM PIP2 is limited. Here, we describe the functions of Nir1 as a positive regulator of Nir2 and PIP2 homeostasis. In contrast to the family proteins Nir2 and Nir3, Nir1 constitutively localizes at ER-PM junctions. Nir1 potentiates Nir2 targeting to ER-PM junctions during receptor-mediated signaling and is required for efficient PM PIP2 replenishment. Live-cell imaging and biochemical analysis reveal that Nir1 interacts with Nir2 via a region between the FFAT motif and the DDHD domain. Combined, results from this study identify Nir1 as an ER-PM junction localized protein that promotes Nir2 recruitment for PIP2 homeostasis.
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