Elicitins trap and transfer sterols from micelles, liposomes and plant plasma membranes

Abstract

Using elicitins, proteins secreted by some phytopathogenic Oomycetes (Phytophthora) known to be able to transfer sterols between phospholipid vesicles, the transfer of sterols between micelles, liposomes and biological membranes was studied. Firstly, a simple fluorometric method to screen the sterol-carrier capacity of proteins, avoiding the preparation of sterol-containing phospholipidic vesicles, is proposed. The transfer of sterols between DHE micelles (donor) and stigmasterol or cholesterol micelles (acceptor) was directly measured, as the increase in DHE fluorescence signal. The results obtained with this rapid and easy method lead to the same conclusions as those previously reported, using fluorescence polarization of a mixture of donor and acceptor phospholipid vesicles, prepared in the presence of different sterols. Therefore, the micelles method can be useful to screen proteins for their sterol carrier activity. Secondly, elicitins are shown to trap sterols from purified plant plasma membranes and to transfer sterols from micelles to these biological membranes. This property should contribute to understand the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions.

Mediation of Elicitin Activity on Tobacco Is Assumed by Elicitin-Sterol Complexes

“…The purified proteins (native state) from both Phytophthora and Pichia culture filtrates are "void," i.e., sterol-unloaded proteins, as it appeared in this work and from structural (Boissy et al, 1996;Fefeu et al, 1997;Gooley et al, 1998) and chemical (Mikes et al, 1997(Mikes et al, , 1998 analyses. It was previously reported that elicitins easily bind sterols from tobacco plasma membranes (Vauthrin et al, 1999). Thus, during ligand replacement and binding experiments elicitins can load sterols.…”

“…The iodination of elicitin changed neither the previously described effects of the elicitor on tobacco cells nor the sterol carrier activity (Osman, Vauthrin, Mikes, Milat, Panabières, Marais, Brunie, Maume, Ponchet, and Blein, unpublished results). Plasma membrane-enriched fractions were obtained as previously reported (Vauthrin et al, 1999). Binding experiments and ligand replacement experiments were carried out as already reported .…”

“…They bind sterols and catalyze their transfer between artificial membranes (Mikes et al, 1998). Moreover, they are able to pick up sterols from plasma membranes (Vauthrin et al, 1999). Natural elicitins interact with sterols showing similar binding characteristics, i.e., a 1:1 sterol: protein stoichiometry, and the dissociation constants are in the same range.…”

“…Natural elicitins interact with sterols showing similar binding characteristics, i.e., a 1:1 sterol: protein stoichiometry, and the dissociation constants are in the same range. Despite these identities, elicitins exhibit differences with regard to the kinetics of loading and the rates of sterol exchanges between liposomes or micelles (Mikes et al, 1998;Vauthrin et al, 1999). The most efficient elicitin is cryptogein (from Phytophthora cryptogea), the less efficient being parasiticein and capsicein, secreted by Phytophthora parasitica and Phytophthora capsici, respectively.…”

Involvement of lipid-protein complexes in plant-microorganism interactions

“…Moreover, elicitins catalyze sterol transfer between phospholipidic artificial membranes [49,50]. They are also able to trap sterols from biological membranes (plant cell suspensions or purified plasma membranes) and to transfer sterols from liposomes to isolated plasmalemma vesicles [51]. In addition, elicitins bind fatty acids and phospholipids.…”