Intracellular localization of a lipid transfer protein in Vigna unguiculata seeds Andre´ de O. Carvalho a , Carlos Eduardo de S. Teodoro a , Maura Da Cunha b , Anna L. Okorokova-Fac¸anha a , Lev A. Okorokov a , Ka´tia V. S. Fernandes c and Valdirene M. Gomes a, * a Laborato ´rio de Fisiologia e Bioquı´mica de Microrganismos, Centro de Biocieˆncias e Biotecnologia, Universidade Estadual do Norte Fluminense, Avenida Alberto Lamego 2000, 28013–600, Campos dos Goytacazes, RJ, Brazil b Laborato ´rio de Biologia Celular e Tecidual, Centro de Biocieˆncias e Biotecnologia, Universidade Estadual do Norte Fluminense, Avenida Alberto Lamego 2000, 28013–600, Campos dos Goytacazes, RJ, Brazil c Laborato ´rio de Quı´mica e Func ¸a ˜o de Proteı´nas e Peptı´deos, Universidade Estadual do Norte Fluminense, Avenida Alberto Lamego 2000, 28013–600, Campos dos Goytacazes, RJ, Brazil *Corresponding author, e-mail: valmg@uenf.br Received 25 May 2004; revised 13 July 2004 Lipid transfer proteins (LTP) facilitate transfer of lipids between membranes in vitro. Up to now, they have been found to be localized basically in the plant cell wall and in compartments linked to lipid metabolism, such as glyoxy- somes. Accordingly, LTP are considered to be involved in the plant defence against pathogen microbes and lipid metab- olism. We herein show, by immunoelectron microscopy, that besides the cell wall, LTP are localized in the lumen of organelles which we suggest to be the protein storage vacuoles, as well as in vesicles similar to the lipid-containing ones and in the extracellular space of Vigna unguiculata seeds. To further characterize these organelles, we performed subcellular fractionation of membranes isolated from imbibed seeds on a sucrose-density gradient. The analysis of these fractions revealed that the lightest membrane vesicles, derived probably from PSV, contain LTP, a-TIP and K 1 independent PP i ase, but not g -TIP and K 1 stimulated PP i ase. The pre- sence of LTP and vicilins (typical storage protein) in the lumen of these vesicles was confirmed by immunoelectron microscopy. Taken together, the data suggest that the intra- cellular LTP in the V. unguiculata seeds are localized in protein storage vacuoles and in lipid-containing vesicles. Introduction The idea that a membrane biogenesis requires the trans- fer of phospholipids from the site of their synthesis to different cellular membranes led to the discovery of pro- teins that facilitate the exchange of lipids between mem- branes, in vitro (Kader 1975, Arondel and Kader 1990). These-non-specific lipid transfer proteins (LTP) share several common properties: the ability to bind fatty acids and their derivatives, molecular masses of 9–10 kDa, high isoelectric points and the presence of eight cysteine residues engaged in four disulphide bridges (Kader 1996). The ability of LTP to bind hydrophobic molecules (Tsuboi et al. 1992, Sodamo et al. 1997) agrees well with structural studies which have shown that LTP possess four a-helices stabilized by disulphide bounds forming an internal hydrophobic cavity which runs through the protein molecule. It has been also shown that this cavity is able to accommodate hydrophobic molecules of different sizes (Lerche et al. 1997, Lee et al. 1998, Zachowski et al. 1998). The finding of a putative signal peptide in the LTP sequences suggested that the protein could be targeted to a specific intracellular compartment and/or secreted (Vignols et al. 1994). In the latter case, the participation of LTP in lipid metabolism, including the lipid transfer between intracellular membranes, was not obvious, raising the possibility of the existence of at least two populations PHYSIOLOGIA PLANTARUM 122: 328–336. 2004 doi: 10.1111/j.1399-3054.2004.00413.x PrintedinDenmark–allrightsreserved Copyright #PhysiologiaPlantarum2004 Abbreviations – DTT, dithiothreitol; LTP, lipid transfer protein; MOPS, 3-[N-morpholino] propane-sulphonic acid; PMSF, phenylmethylsufonylfluoride; PP i ase, vacuolar H 1 translocating inorganic pyrophosphatase; PSV, protein storage vacuole; PVPP, polyvinylpolypyrrolidone; TIP, tonoplast intrinsic protein. 328 Physiol. Plant. 122, 2004