Diacylglycerol Induces Fusion of Nuclear Envelope Membrane Precursor Vesicles

Banafshe Larijani,Richard D. Byrne,Teresa Barona,Dominic L. Poccia,Michael J.O. Wakelam,Trevor R. Pettitt

doi:10.1074/jbc.m412863200

Abstract

Purified membrane vesicles isolated from sea urchin eggs form nuclear envelopes around sperm nuclei following GTP hydrolysis in the presence of cytosol. A low density subfraction of these vesicles (MV1), highly enriched in phosphatidylinositol (PtdIns), is required for nuclear envelope formation. Membrane fusion of MV1 with a second fraction that contributes most of the nuclear envelope can be initiated without GTP by an exogenous bacterial PtdIns-specific phospholipase C (PI-PLC) which hydrolyzes PtdIns to form diacylglycerides and inositol 1-phosphate. This PI-PLC hydrolyzes a subset of sea urchin membrane vesicle PtdIns into diglycerides enriched in long chain, polyunsaturated species as revealed by a novel liquid chromatography-mass spectrometry analysis. Large unilammelar vesicles (LUVs) enriched in PtdIns can substitute for MV1 in PI-PLC induced nuclear envelope formation. Moreover, MV1 prehydrolyzed with PI-PLC and washed to remove inositols leads to spontaneous nuclear envelope formation with MV2 without further PI-PLC treatment. LUVs enriched in diacylglycerol mimic prehydrolyzed MV1. These results indicate that production of membrane-destabilizing diglycerides in membranes enriched in PtdIns may facilitate membrane fusion in a natural membrane system and suggest that MV1, which binds only to two places on the sperm nucleus, may initiate fusion locally.

Highlights

Male pronuclear or somatic nuclear envelope formation involves binding of nuclear membrane precursors to the chromatin surface followed by fusion to create a double membrane enclosing the chromatin [1, 5,6,7]
We have previously reported that envelope formation in a cell-free system derived from sea urchin eggs requires the fusion of three egg membrane vesicle populations and remnants of the sperm nuclear envelope at the tip and base of the conical nucleus (8 –10)
MV1 binds at the tip and base of the sperm nucleus and is required for nuclear envelope formation, which can be induced by addition of GTP or a bacterial PtdIns-specific phospholipase C (PI-PLC) [9, 12]

Summary

Introduction

Male pronuclear or somatic nuclear envelope formation involves binding of nuclear membrane precursors to the chromatin surface followed by fusion to create a double membrane enclosing the chromatin [1, 5,6,7]. MV1 binds at the tip and base of the sperm nucleus and is required for nuclear envelope formation, which can be induced by addition of GTP or a bacterial PtdIns-specific phospholipase C (PI-PLC) [9, 12]. PtdIns hydrolysis is best known as an intermediate step in G-protein signaling pathways in which PtdIns[4,5]P2 is hydrolyzed by PI-PLC to form diacylglycerol (DAG) and inositol-1,4,5 triphosphate (InsP3). Such signaling occurs in membranes containing 3–10% PtdIns with much lower amounts of PtdIns[4,5]P2 [15]. DRG, diradylglyceride; PtdIns, phosphatidylinositol; PtdCho, phosphatidylcholine; InsP3, inositol 1,4,5-triphosphate; PI-PLC, PtdIns-specific phospholipase C; LC-MS, liquid chromatography-mass spectrometry; LUV, large unilammelar vesicle; GTP␥S, 5-guanosine-5Ј-(␥-thio)triphosphate; ET-18-OCH3 or ET, 1-O-octadecyl-2-O-methylsn-glycero-3-phosphorylcholine; LB, egg lysis buffer; S150, cytosolic egg extract

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Conclusion