EFFECT OF SINGLE-CHAIN LIPIDS ON PHOSPHOLIPASE C-PROMOTED VESICLE FUSION - A TEST FOR THE STALK HYPOTHESIS OF MEMBRANE-FUSION

The effect of low proportions (up to 5 mol %) of single-chain lipids o n phospholipase C-promoted fusion of large unilamellar vesicles has be en investigated with the aim of testing the so-called stalk model of m embrane fusion. This model is known in two main versions, the one orig inally published by Kozlov and Markin [Kozlov, M. M. and Markin, V. S. (1983) Biofizika 28, 255-261] and what is known as the ''modified sta lk model'' [Siegel, D. P. (1993) Biophys. J. 65, 2124-2140], that diff er in a number of predictions. In the view of the latter author, hydro carbons or other nonpolar lipids should help fusion by decreasing the interstitial energy of the stalk connecting the two apposed bilayers. We show that small amounts of hexadecane or squalene increase signific antly the fusion rates in our system. Changes in monolayer curvature a re the object of different predictions by the original and modified st alk theories. According to the original form, fusion would be promoted by lipids inducing a negative curvature in the closest (cis) monolaye rs of the fusing membranes and inhibited by the same lipids in the tra ns monolayers; the opposite would happen with lipids inducing a positi ve curvature. The modified stalk model predicts that fusion is helped by increasing the negative curvature of both monolayers. In our system , symmetrically distributed arachidonic acid, which increases the nega tive curvature, enhances lipid and content mixing, and the opposite is found with symmetrically distributed lysophosphatidylcholine or palmi toylcarnitine, which facilitate a positive monolayer curvature. In add ition, fluorescence polarization and P-31 NMR studies of the lamellar- to-isotropic (Q(224) cubic) thermotropic transition of a lipid mixture corresponding to our liposomal composition reveal that all lipids tha t facilitate fusion decrease the transition temperature, while fusion inhibitors increase the transition temperature. Moreover, fusion (cont ent mixing) rates show a maximum at the lamellar-to-isotropic transiti on temperature. These observations support the involvement of inverted lipid structures, as occurring in the inverted cubic phases, in membr ane fusion. All these data are in full agreement with the stalk model of membrane fusion, particularly in its modified version.