Purification and characterization of a spinach-leaf protein capable of transferring phospholipids from liposomes to mitochondria or chloroplasts

Abstract

A phospholipid transfer protein has been purified 195-fold from an extract of spinach leaves. This protein is capable of transferring phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol and phosphatidylethanolamine from liposomes to mitochondria. In addition to this protein, a minor part of the total activity was associated with a less purified fraction.The pure protein has an isoelectric point of 9.0 5 0.2 determined by a chromatofocusing technique.Electrophoresis on sodium dodecyl sulfate/polyacrylamide gel showed that the protein is homogeneous and has an apparent molecular weight of 9000 1000, in agreement with the value (8832) calculated from the amino acid composition. This composition is characterized by a high amount of alanine and glycine and by the absence of phenylalanine, whereas arginine, glutamine, histidine and methionine are minor components.The spinach protein is also able to transfer phosphatidylcholine and phosphatidylglycerol from liposomes to intact chloroplasts. This observation reinforces the hypothesis that plastid phospholipids are partly imported from outside the organelle by a transfer process.The presence of phospholipid traqsfer proteins has been established in several plant tissues, including potato tuber [l], castor bean [2-51, maize seeds [6]. The properties of these proteins were compared to those phospholipid transfer proteins highly purified from animal sources [7-171. Since all these proteins are able to transfer phospholipids from liposomes or microsomes to mitochondria, it has been suggested that phospholipid transfer proteins participate in the biogenesis of mitochondria1 membranes [18 -221. In order to examine whether this hypothesis is also valid with chloroplasts, investigations have been carried out on photosynthetic tissues, particularly on spinach leaves which are a suitable material for the isolation of chloroplasts. A phosphatidylcholine transfer activity involving mitochondria and chloroplasts has been detected in homogenates prepared from spinach or Avena leaves [23,24]. Howver, the occurrence of these proteins in spinach leaves was questioned in investigations carried out with crude extracts [25]. The presence of interfering enzymes (proteases, lipases), which can be eliminated by gel filtration, explains this discrepancy [24]. It remained to further purify the active spinach protein. In preliminary studies, presented in two short reports [26,27], we have found that two basic phospholipid transfer proteins are present in spinach leaf cytosol. In the present paper, we report the purification and the characterization of a phospholipid transfer protein from spinach leaves. MATERIALS AND METHODSSephadex G-75, Sephadex G-50 and CM-Sepharose C1-6B were obtained from Pharmacia (Sweden). DEAE-Trisacryl This is paper no. 12 in the series: Proteins and the Intracellular Exchange of Lipids. Preparation of intact organellesMitochondria were isolated from potato tubers by sucrosegradient centrifugation [2] and stored in 10 mM Tris/HCI, 250 mM sucrose, 1 mM Na4ED...

Comparative study of the lateral motion of extrinsic probes and anthracene‐labelled constitutive phospholipids in the plasma membrane of Chinese hamster ovary cells

“…FRAP experiments were therefore performed with CHO cells exogenously labelled with a synthetic anthracene-phosphatidylcholine (acylANnoGroPCho), using a phospholipid exchange protein of plant origin [18]. Under the conditions used [18], this phospholipid can be assumed to be localized in the external leaflet of the plasma membrane. The lateral diffusion coefficient measured for it (Table 1) was slightly lower than that obtained for the anthracene-phospholipids after metabolic incorporation of ANno.…”

“…250 p1 sonicated lipid suspension was mixed with 1 ml cell suspension (3 x 106 cells/ml). 100 p1 buffer containing the phospholipid exchange protein (500 pg/ml) purified from spinach leaves [18] was then added. Incubation was carried out for 15 min at 37°C.…”

On the role of a Lipid-Transfer Protein. Arabidopsis ltp3 mutant is compromised in germination and seedling growth.

“…[1][2][3][4][5] Unlike their animal counterparts, plant LTPs typically have broad substrate specificity and are also named nonspecific LTPs. Their affinity for lipids is presumed to be crucial for their biological function, but in vivo substrates remain largely unknown.…”

“…LTPs were identified according to their ability to bind phospholipids and fatty acids and were originally proposed to participate in the intracellular transfer of lipids. 2 These peptides present a common structural feature which includes 8 cysteine residues involved in 4 disulfide bridges and a consensus amino acid signature (Prosite PS00597). Several LTP structures have been determined and revealed the presence of 4 a helices which define a central hydrophobic cavity that can accommodate lipids and justifies their lipid-binding properties.…”

A 10 kD barley basic protein transfers phosphatidylcholine from liposomes to mitochondria

“…Tissues from other plants such as maize endosperms plus embryos as well as coleoptiles ( [3H]-phosphatidylcholine (0.3 Ci x tool ~) was prepared from potato tuber slices incubated with methyl-jail] -choline chloride as previously described (5). Maize phospholipid transfer protein was purified from three day-old seedlings by CMSepharose chromatography as previously described (3).…”