Vesicle Membrane Fusion Induced by the Concerted Activities of Sphingomyelinase and Phospholipase C*
- From the Grupo Biomembranas (Unidad Asociada al C.S.I.C.), Departamento de Bioquı́mica, Universidad del Paı́s Vasco, Aptdo. 644, 48080 Bilbao, Spain
Abstract
When vesicles composed of an equimolar mixture of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with phospholipase C, phospholipid hydrolysis occurs without major changes in vesicle architecture. In the same way, addition of sphingomyelinase leads only to sphingomyelin cleavage. However, when both enzymes are added together, their joint hydrolytic activities give rise to leakage-free vesicle aggregation, lipid mixing, and aqueous contents mixing, i.e. vesicle fusion. The contribution of both enzymes is unequal, the main role of sphingomyelinase being the production of relatively large amounts of ceramide that will facilitate the lamellar-to-nonlamellar transition in the formation of the fusion pore, whereas phospholipase C provides mainly a localized, asymmetric, high concentration of diacylglycerol that constitutes the trigger for the fusion process. The lipidic end-products of both enzymes cooperate in destabilizing and fusing the membranes in a way that is never achieved through the action of any of the enzymes individually, nor by the products themselves when premixed with the other lipids during liposome preparation. Thus the enzymes appear to be coupled through their reaction products. This is the first observation of membrane fusion induced by the concerted activities of two enzymes. Besides, considering that both diacylglycerol and ceramide are important metabolites involved in cell signaling, it may also provide new ideas in the exploration of “cross-talk” phenomena between different signal transduction pathways.
Footnotes
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↵* This work was supported in part by funds from Dirección General de Investigación Cientifica y Técnica (Grant PB96–0171) and the Basque Government (Grant PI96/46).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom reprint requests should be addressed. Tel.: 34-94-4647700, ext. 2407; Fax: 34-94-4648500; E-mail: gbpaliza{at}lg.ehu.es.
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↵2 M. B. Ruiz-Argüello, unpublished results.
- Abbreviations:
- PC
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phosphatidylcholine
- Ch
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cholesterol
- LUV
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large unilamellar vesicles
- PE
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phosphatidylethanolamine
- SM
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sphingomyelin
- ANTS
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8-aminonaphthalene-1,3,6-trisulfonic acid
- DPX
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p-xylenebis (pyridinium bromide).
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- Received December 9, 1997.
- Revision received June 8, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
