RDGBα localization and function at membrane contact sites is regulated by FFAT–VAP interactions

Abstract

Phosphatidylinositol transfer proteins (PITPs) are essential regulators of PLC signalling. The PI transfer domain (PITPd) of multi-domain PITPs is reported to be sufficient for function, questioning the relevance of other domains in the protein. In photoreceptors, loss of RDGBα, a multi-domain PITP localized to membrane contact sites (MCSs), results in multiple defects during PLC signalling. Here, we report that the PITPd of RDGBα does not localize to MCSs and fails to support function during strong PLC stimulation. We show that the MCS localization of RDGBα depends on the interaction of its FFAT motif with dVAP-A. Disruption of the FFAT motif (RDGB) or downregulation of dVAP-A, both result in mis-localization of RDGBα and are associated with loss of function. Importantly, the ability of the PITPd in full-length RDGB to rescue mutant phenotypes was significantly worse than that of the PITPd alone, indicating that an intact FFAT motif is necessary for PITPd activity Thus, the interaction between the FFAT motif and dVAP-A confers not only localization but also intramolecular regulation on lipid transfer by the PITPd of RDGBα. This article has an associated First Person interview with the first author of the paper.

Extended synaptotagmin regulates plasma membrane-endoplasmic reticulum contact site structure and lipid transfer functionin vivo

“…Thus our data clearly support a role 395 for dEsyt in supporting the lipid transfer function of RDGB at ER-PM contact sites in vivo. 396 Consistent with this model, we found that the dEsyt protein is localized to the base of the 397 microvillar PM at the ER-PM contact site where the RDGB protein is also specifically 398 localized (Vihtelic et al, 1993;Yadav et al, 2018). 399 400 Although we found a normal density of ER-PM MCS in dEsyt KO photoreceptors at eclosion, 401…”

Regulation of PI4P levels by PI4KIIIα during G-protein-coupled PLC signaling in Drosophila photoreceptors

“…Through this method, we determined the levels of PIP and PIP 2 in retinal samples. As a positive control, we studied the levels of PI, PIP and PIP 2 in retinae from rdgB mutants; rdgB encodes an eye-enriched phosphatidylinositol transfer protein (Trivedi and Padinjat, 2007) that is required to support PI(4,5)P 2 resynthesis during phototransduction (Hardie et al, 2001(Hardie et al, , 2015Yadav et al, 2015Yadav et al, , 2018. Using our assay, we found that the levels of 36:2 and 36:3 species of PIP and PIP 2 , which were the most abundant species in retinal tissue ( Fig.…”

Sticking With It: ER-PM Membrane Contact Sites as a Coordinating Nexus for Regulating Lipids and Proteins at the Cell Cortex

“…Phospholipid exchange occurs between ER and the PM at RdgBα-enriched ER-PM MCSs ( Cockcroft and Raghu, 2016 ; Cockcroft et al, 2016 ). The clustering of RdgBα at these ER-PM contact sites involves its FFAT motif, which enables RdgBα recruitment by Drosophila VAP-A ( Yadav et al, 2018 ). Moreover, the absence of VAP-A leads to RdgBα mis-localization, preventing it from performing its function at ER-PM MCSs thereby disrupting phototransduction ( Yadav et al, 2018 ).…”