Requirements for biomembrane generation. Restitution of ER and nuclear membranes is accomplished by site-specific membrane biogenesis. and is not derived from retrograde transport of lipids, membrane rafts or caveolea from cell membrane. (A) Incubation of ER, IN, ONM with active CC enriched with ceramide-labeled sphingolipids isolated from Golgi vesicles (A-1, B-1), cell membrane rafts and caveolea (A-2, B-2) and cell apical membrane (A-3, B-3), has not produced the membranes labeled with sphingolipids. The lipid extracts from incubated ER, IN, ONM (A1-3), respectively were free of radiolabeled lipids provided in CC. The labeled sphingolipids from Golgi vesicles and membrane rafts (A4) and from apical membranes (A5) remained in the CC. The incubation of INM with nuclear contents enriched with radiolabeled precursors of membrane phospholipids consisting [³H] arachidonate and [³H]inositol has not generated radiolabeled INM (A-6). In contrast, the active CC enriched with radiolabeled arachidonate, and inositol generated radiolabeled membranes of IN (A-7), ONM (A-8), INM (A-9), ER (A-10) and ER transport vesicles (A-11). The lipid extracts from the membranes (IN, ONM, INM, ER, ER transport vesicles), from CC and nuclear contents were applied to HPTLC and scanned in Berthold Analyzer for 16 hours. The [³H]palmitate labeled ceramides and sphingolipids prepared from Golgi vesicles (A-1) cell membrane rafts and caveolea (A-2) and cell apical membrane (A-3) as depicted in B-1-3 were used. The amount of radiolabeled lipids added to CC corresponds to the one depicted in B. The lipid samples shown in A represents material applied to HPTLC and subjected to chromatography in the solvent system separating free fatty acids (ethyl ether/hexanes, 7:3, v/v), while sphingolipids, phosphosphingolipids and phosphoglycerides remain unseparated and close to the origin. The detection of radiolabeled lipid was performed in Berthold radioactivity analyzer for 16 h. (B) [³H]palmitate labeled sphingolipids corresponding to the amount used in incubation described in A following high performance thin layer chromatography (HPTLC) in chloroform/methanol/ water (65:35:8, v/v/v) mixture and scanning in Berthold analyzer for 16 h. The lanes correspond to sphingolipids extracted from preparations of 1-Golgi vesicles, 2-apical membrane rafts and caveolea, 3-apical membranes. (C) Synthesis of membrane lipids is affected by degradation of RNA in active CC. Synthesis of arachidonate and inositol-labeled lipids in IN, ONM, ER (C1-3) respectively, in the presence of RNase treated (C1-3), and IN, ONM in untreated (C-4, 5) CC. The scanning of the radiolabeled membranes was performed on membrane lipid extracts applied to HPTLC as described in A and scanned for 16 h in Berthold Analyzer. (D) The [³H]inositol labeled lipids of IN (D-1), ONM (D-2), INM (D-3) are more complex than those synthesized in ER (D-4). HPTLC of the lipid extracts was performed in chloroform/methanol/acetic acid/water, 65:35:8:4, by vol.) and scanned in Berthold radioactivity analyzer for 16 h.

Interaction of NIP-labeled 30kDa protein of CC with lipids of IN, ONM, and INM represented by phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidic acid (PA) and sphingolipids of ER-transport vesicles and Golgi represented by ceramides (Cer) and sphingomyelin (SM) and neutral lipids (NL) consisting of glycerides and cholesterol. Microtiter plates were coated with of with 0.5 to 20 μg lipids extracted from the IN, ONM, INM, ER transport vesicles, Golgi transport vesicles and cell apical membrane, reacted with 1.0-1.5 A₂₈₀ NIP-labeled 30kDa cytosolic protein for 120 min, followed by incubation with CC proteins (1mg/ml). After incubation, the plates were rinsed with PBS containing 0.05% Tween and read at A₂₈₀. Each reaction was performed in triplicate.

Incubation of IN in active cytosol (CC) demonstrates movement of membrane lipids from ONM to INM and changes in phosphatidylinositides profile. During 60 min incubation with CC, the radiolabeled lipids decrease in the ONM and INM (A and B, respectively). The IN labeled with [³H]arachidonate and [³H]inositol were subjected to incubation with unlabeled CC, followed by inner and outer membrane separation and detection of the relative amount of radiolabeled lipid present in the ONM and INM of the IN after 30 and 60 min incubation. The level and the initial composition of the labeled phospholipids in ONM (Lane A) and INM (lane B) is depicted in panel C. Panel A demonstrates initial labeling of ONM (1), after 30 min (2) and 60 min (3) incubation of the IN. Panel B demonstrates the initial (1), 30 min (2) and 60 min (3) labeling of INM phospholipids. Following 60 min incubation of the labeled IN with CC, the IN were first subjected to the sucrose gradient purification and then to isolation of the ONM and INM and lipid extraction depicted in panels A and B. The lipids recovered from sucrose gradient-separated material from IN is depicted in panel D. The lipid extracts from the separated membranes were applied to HPTLC and subjected to two dimensional thin layer chromatography in solvent system consisting of chloroform/methanol/ammonia, 80:35:5, v/v/v, and after thorough drying to second dimension chromatography in chloroform/acetone/methanol/acetic acid/ water 30:40;10:10:5, by vol. The plates were dried and subjected to 16-h counting in Berthold Radioactivity Analyzer, to phospholipids detection, carbohydrate detection, ninhydrin-positive phospholipids detection and sulfuric acid charring. The combination of the screening allowed us to identify individual lipid of the membranes shown in panel D that consisted of phosphatidylcholine (PC) phosphatidylinositol (PI) and phosphatidic acid (PA).

Phosphatidylinositides, and phosphoglycerides comprised of phosphatidylcholine (PC) and phosphatidic acid generate ONM and INM of nuclear membranes. The two dimensional HPTLC was used to identify phospholipids of ONM (A), IN (B), INM (C), ER (D). The HPTLC was fist performed in solvent mixture consisting of chloroform/methanol/acetone/25% ammonium hydroxide 43:38:5:8, by vol. dried thoroughly to remove excess of moisture and ammonia and developed in second dimension in solvent system consisting of chloroform/methanol/acetic acid/water (65:35:8:4, by vol.). To identify different phospholipids, the HPTLC were subjected to iodine vapors, ninhydrin spray, phospholipids detecting spray and orcinol-carbohydrate-detecting spray. Based on the results of these experiments the lipids isolated from the membranes were identified as 1-phosphatidylinositol bisphosphate (PIP2), 1'-phosphatidylinositol monophosphate (PIP), 1''-phosphatidylinositol monophosphate (PIP), 2-phosphatidylcholine (PC), 3-phosphatidylcholine (PC), 4-phosphatidylinositol (PI), 5-phosphatidic acid (PA).

Identification of 30 kDa CC protein in IN, ONM, INM following incubation with NIP-labeled CC protein is depicted in the upper panel. The protein extracts from IN (lanes 1,4) ONM (lanes 2,5) and INM (lanes 3,6) incubated with NIP-CC protein for 30 min (lanes 1-3) and 60 min (lanes 4-6) and the purified 30 kDa NIP-protein (lane 7) were subjected to SDS-PAGE and western blot. The NIP labeled proteins retained on the membranes were identified with anti NIP IgG.