PLCγ is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly

Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MVI) required for NE assembly has a unique lipid,composition consisting prominently of poly-phosphatidylinositides. The lipid composition was determined by adapting HPLC electrospray ionisation tandem mass spectrometry to phosphoinositide analysis, revealing the capacity of this technique to document dynamic lipid transitions of functional importance in natural mernbrane populations. MV1 is > 100-fold enriched in endogenous PLC gamma and > 25-fold enriched in the PLC substrate phosphatidylinositol bisphosphate (PtdInsP(2)) compared to the second membrane population, derived largely from endoplasmic reticulum (ER), that contributes most of the NE. During NE fort-nation PLC gamma becomes transiently phosphorylated at the tyrosine 783 site indicative of its activation. In addition specific inhibition of PLC gamma blocks nuclear envelope formation. In vivo, PLC-gamma is concentrated on vesicles of similar size to purified MV1. These associate with nuclei during the period of NE formation and are distinct from ER membranes. The unprecedented concentration of PLC-gamma and its substrate PtdInsP(2) in a subset of membranes that binds to only two regions of the nucleus, and activation of PLC gamma by GTP during initial stages of NE formation provide a mechanism for temporal control of NE assembly and offer an explanation for how such a process of membrane fusion can be spatially regulated. (c) 2006 Elsevier Inc. All rights reserved.