Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum

In the purple membrane (PM) of Halobacterium salinarum is present a phospholipid dimer consisting of sulfo-triglycosyl-diether (S-TGD-1) esterified to the phosphate group of phosphatidic acid (PA), i.e., S-TGD-1-PA, called glycocardiolipin (GlyC) (Corcelli, A., M. Colella, G. Mascolo, F. P. Fanizzi, and M. Kates. A novel glycolipid and phospholipid in the purple membrane. 2000. Biochemistry. 39: 3318–3326). The GlyC content of whole cells, PM, and other cell fractions of H. salinarum have been analyzed. GlyC is a nonabundant phospholipid in H. salinarum cells, and it represents one of the major phospholipids of isolated PM.In this report, we show that a) GlyC is formed during the isolation of PM, b) GlyC increase in H. salinarum cells is specifically induced by osmotic shock, and c) in correspondence with GlyC increase, a decrease of S-TGD-1 levels occurs. The changes in membrane lipid composition observed during the isolation of PM are due to de novo synthesis of GlyC from S-TGD-1. In the purple membrane (PM) of Halobacterium salinarum is present a phospholipid dimer consisting of sulfo-triglycosyl-diether (S-TGD-1) esterified to the phosphate group of phosphatidic acid (PA), i.e., S-TGD-1-PA, called glycocardiolipin (GlyC) (Corcelli, A., M. Colella, G. Mascolo, F. P. Fanizzi, and M. Kates. A novel glycolipid and phospholipid in the purple membrane. 2000. Biochemistry. 39: 3318–3326). The GlyC content of whole cells, PM, and other cell fractions of H. salinarum have been analyzed. GlyC is a nonabundant phospholipid in H. salinarum cells, and it represents one of the major phospholipids of isolated PM. In this report, we show that a) GlyC is formed during the isolation of PM, b) GlyC increase in H. salinarum cells is specifically induced by osmotic shock, and c) in correspondence with GlyC increase, a decrease of S-TGD-1 levels occurs. The changes in membrane lipid composition observed during the isolation of PM are due to de novo synthesis of GlyC from S-TGD-1. The finding of cardiolipin analogs in the extreme halophilic Archaea arises from an investigation into the characteristics of delipidated bacteriorhodopsin (BR), the photoactivated proton pump in the purple membrane (PM) of Halobacterium salinarum (1Lopez F. Lobasso S. Colella M. Agostiano A. Corcelli A. Light-dependent and biochemical properties of two different bands of bacteriorhodopsin isolated on phenyl-sepharose CL-4B.Photochem. Photobiol. 1999; 69: 599-604Crossref Scopus (25) Google Scholar). By analyzing lipids of delipidated BR fractions by electrospray ionization-mass spectrometry (ESI-MS), it was found that the composition of the residual lipids in delipidated BR is different from that of the PM in that the delipidated fractions have much higher proportions of two bicharged peaks at 760 m/z and 966 m/z, corresponding to two novel membrane lipids. This suggested that these novel lipids are more resistant to detergents than the other lipid components of PM, or, alternatively, that the novel lipids are more strongly bound to BR and may be involved in stabilizing the BR trimer structure. The two novel PM lipids were isolated, purified by thin-layer chromatography (TLC), and analyzed by conventional analytical and spectroscopic methods to determine their chemical structure. Combining ESI-MS data and proton and phosphorus nuclear magnetic resonance (NMR) data of the purified novel lipids, together with the identification of their acid degradation products, allowed the elucidation of the chemical structures of the novel PM phospholipids (2Corcelli A. Colella M. Mascolo G. Fanizzi F.P. Kates M. A novel glycolipid and phospholipid in the purple membrane.Biochemistry. 2000; 39: 3318-3326Crossref PubMed Scopus (73) Google Scholar). Their structures are, respectively, of a phosphosulfoglycolipid, 3-HSO3-Galpβ1-6Manpα1-2Glcpα-1-1-[sn-2,3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycerol] (called glycocardiolipin, or GlyC), and a glyceroldiether analog of bisphosphatidylglycerol, sn-2,3-di-O-phytanyl-1-phosphoglycerol-3-phospho-sn-2,3-di-O-phytanylglycerol (archaeal BPG) (Fig. 1). Both novel lipids have two diphytanylglycerol moieties in their molecule, making their dimeric structures analogous to that of eukaryal cardiolipin. Interestingly, GlyC consists of a sulfotriglycosyldiphytanylglycerol esterified to the phosphate group of phosphatidic acid (PA); in addition, its polar head group is composed of the same sugars in the same sequence and anomeric configuration as in sulfo-triglycosyl-diether-1 (S-TGD-1), the major membrane glycolipid of Halobacterium, namely βGalp→αMan→αGlc. Also, the sulfate group is located on C-3 of the galactose residue, as in S-TGD-1 (3Kates M. Deroo P.W. Structure determination of the glycolipid sulfate from the extreme halophile Halobacterium cutirubrum.J. Lipid Res. 1973; 14: 438-445Abstract Full Text PDF PubMed Google Scholar). We will refer to it as S-TGD-1-PA (see structure in Fig. 1A) or GlyC, adopting a previously introduced abbreviation (4Corcelli A. Lattanzio V.M.T. Mascolo G. Papadia P. Fanizzi F.P. Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane.J. Lipid Res. 2002; 43: 132-140Abstract Full Text Full Text PDF PubMed Google Scholar). The cardiolipin/BR stoichiometries in the PM have also been estimated; one GlyC per BR is present in the PM, while BPG is only a minor component (4Corcelli A. Lattanzio V.M.T. Mascolo G. Papadia P. Fanizzi F.P. Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane.J. Lipid Res. 2002; 43: 132-140Abstract Full Text Full Text PDF PubMed Google Scholar). Here, by analyzing the distribution of GlyC in the various fractions obtained in the course of PM isolation, we show that GlyC is present in very low amounts in the cells of H. salinarum under physiological conditions, and that it is de novo synthesized and accumulated mainly in the PM during cell lysis by osmotic shock. DNase I was obtained from Sigma. All organic solvents used were commercially distilled and of the highest available purity (Sigma-Aldrich). TLC plates (silica gel 60A), obtained from Merck, were washed twice with chloroform-methanol (1:1; v/v) and activated at 120°C before use. The engineered high-producing BR strain of H. salinarum used in this study was kindly provided by Richard Needleman (5Ni B.F. Chang M. Duschl A. Lanyi J.K. Needlemann R. An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium.Gene. 1990; 90: 169-172Crossref PubMed Scopus (114) Google Scholar); the H. salinarum NRC-1 strain was kindly provided by Aharon Oren. The H. salinarum cells were grown in light at 37°C in liquid growth medium containing neutralized peptone (L34, Oxoid) prepared as previously described (6Oesterhelt D. Stoeckenius W. Isolation of cell membrane of Halobacterium halobium and its fractionation into red and purple membrane.Methods Enzymol. 1974; 31: 667-678Crossref PubMed Scopus (1592) Google Scholar). PMs were isolated from the high-producing BR strain as previously described (6Oesterhelt D. Stoeckenius W. Isolation of cell membrane of Halobacterium halobium and its fractionation into red and purple membrane.Methods Enzymol. 1974; 31: 667-678Crossref PubMed Scopus (1592) Google Scholar). Briefly, a dialysis bag (cutoff 12,000–14,000) was filled with a concentrated suspension of halobacterial cells in 4 M NaCl in the presence of DNase I and left in water under stirring at 4°C overnight; the purple lysate was centrifuged at 28,000 g for 40 min, and the colorless supernatant decanted. The purple pellet was resuspended in 0.1 M NaCl and again centrifuged; this was repeated two more times, and the last pellet (resuspended in distilled water) was layered over a step gradient (60%, 35%, and 15% sucrose) and then centrifuged at 100,000 g for 18 h at 10°C. The purple band was collected, and sucrose was removed by dialysis. Finally, the collected PMs were suspended in water and frozen (−20°C). During PM isolation, samples of various cellular fractions were stored to be extracted (whole cells, lysed cells after dialysis, supernatant after the first centrifugation, and PM). A suspension of cells (in 4 M NaCl) was passed through a French pressure cell at 2,000 psi three times and left overnight at 4°C. To remove salt from cells broken by French press, an aliquot of a previous sample was dialyzed in the presence of DNase I against water at 4°C overnight. Cells suspended in 4 M NaCl were dialyzed against water at 4°C for different incubation times (15, 30, and 60 min and 12 h). At intervals, lipids were extracted from equivalent aliquots of the lysate by the standard procedure (see below). The weights of the lipid extracts from these aliquots were not significantly different. A suspension of high-producing BR or NRC-1 cells in 4 M NaCl was diluted 5-fold with a hypo-osmotic solution (0.1 M NaCl) and incubated at 25°C under stirring. At intervals, equivalent aliquots of suspension were removed for determination of osmotic fragility (Abs700 nm) and the lipid extraction. Total lipids were extracted using the Bligh and Dyer method, as modified for extreme halophiles (7Kates M. General analytical procedures.in: Dates M. Techniques of Lipidology. 2nd edition. Elsevier Science Publishers, Amsterdam1986: 112-185Google Scholar); the extracts were carefully dried under N2 before weighing. Typically, before lipid extraction, whole cells were suspended in 4 M NaCl, while PMs were in water. In the case of the lipid extraction from PM high salt, 8 mg of PM in water was centrifuged (28,000 g for 40 min) and resuspended in 4 M NaCl (5 ml) before the extraction. For the experiment of lipid re-extract of cells, an aliquot of cell suspension (in 4 M NaCl) was extracted by the standard procedure; then, organic solvents (chloroform-methanol;Δ:3) were again added to the denatured cellular material and incubated at 80°C for 30 min to obtain a re-extract; this procedure was repeated once more, and the two re-extracts were combined. Total lipid extracts were analyzed by TLC on silica gel (20 cm × 10 cm, layer thickness 0.2 mm). Lipids were eluted with Solvent A (chloroform-methanol-90% acetic acid; 65:4:35) and unless otherwise specified, detected by spraying with sulfuric acid in water, followed by charring at 120°C for 45 min. The quantitative analyses of GlyC content were performed by video densitometry, using the software ImageJ (http://rsb.info.nih.gov/ij). The lipid standard curves were linear in the concentration range of 1–10 μg. For ESI-MS analyses, dried samples of lipid extract were dissolved in chloroform-methanol (1:1). Electrospray mass spectra were obtained with an API 165 mass spectrometer (Applied Biosystems/MSD Sciex, Concord, Ontario, Canada) equipped with a Turboion Spray interface. The samples were analyzed by loop injection introducing, by a 7125 Rheodyne valve, 5 μl of sample into a 25 μl/min flow of chloroform-methanol delivered by a Harvard model 11 syringe pump (South Natick, MA). The instrumental conditions were as follows: nebulizer gas flow (air), 1.2 l/min; curtain gas flow (nitrogen), 1.2 l/min; needle voltage, 5,600 V; interface temperature, ambient; orifice voltage, −150 V; ring voltage, −200 V; mass range, 50–2,000 amu; mass step, 0.1 amu; dwell time, 0.2 ms. With the orifice voltage used, CID-MS spectra were obtained showing [M-H]- and [M-2H]2-parent ions as well as some fragmentation ions. To investigate the location and distribution of GlyC in the H. salinarum cells, we studied the distribution of total cell lipids and GlyC in the course of cell fractionation. Table 1 reports the total lipid content and the lipid/BR ratio of the various fractions obtained in the course of PM isolation by following the fractionation protocol of the H. salinarum cells, originally developed by Oesterhelt and Stoeckenius (6Oesterhelt D. Stoeckenius W. Isolation of cell membrane of Halobacterium halobium and its fractionation into red and purple membrane.Methods Enzymol. 1974; 31: 667-678Crossref PubMed Scopus (1592) Google Scholar). We found that a) ∼25% of lipid material was lost during the dialysis, b) 40% of lipids remained in the clear colorless supernatant of the first centrifugation, and c) PM lipids represented only ∼30% of total cell lipids; at the same time, 70% of starting BR was typically recovered in PM after sucrose gradient.TABLE 1Lipid recovery and lipid/BR ratio during PM isolationLipidLipid/BR%w/wCells100 1Lysed cells75 not PM, purple membrane. BR is in the the lipid/BR ratio was in a not PM, purple membrane. BR is in the the lipid/BR ratio was the TLC of the lipid extracts of the whole cells of H. of lysed cells after dialysis, of PM, and of other membrane present in the be that GlyC is lipids extracted from the whole cells, while it is present in lysed cells after dialysis against water, in PM, as well as in the cell material left in the first in Fig. show the ESI-MS analyses of the lipid extract of whole cells and in with data in Fig. it be that the for GlyC at 966 m/z is in the ESI-MS of the lipids, while it is much higher in the ESI-MS of PM lipids. The in the cell GlyC content before and after the osmotic on the that GlyC is not from the whole cells a to the been previously that the of cardiolipin extraction from may be significantly by the conditions H. in the extraction of cells by PubMed Scopus Google a major phospholipid of that is not PubMed Scopus Google Scholar). cells of H. salinarum were suspended in an 4 M NaCl while lysed cells after dialysis or PM were in medium containing very low or salt, we the that the low content of GlyC in the cell lipid extract on a low of cardiolipin extraction from the cell in the presence of high we have a) and analyzed lipids from the denatured after the first lipid extraction of the cells resuspended in medium to for the presence of GlyC residual lipids, and b) the lipid extraction of PM resuspended in 4 M NaCl a lipid extract a of or is TLC analyses in Fig. that GlyC is in the re-extract (Fig. and present in the extract from PM in high salt (Fig. the after the cell was and lipids were dissolved in and analyzed by GlyC was found in this last residual lipid not the of the cells before lipid extraction, of the used, the recovery of GlyC in the lipid we have analyzed total lipids extracted from H. salinarum cells by French TLC in Fig. that GlyC levels in the total lipid extract of cells by French are very low and with in the extracts of the whole cells after French were dialyzed to low and lipids were extracted after salt that GlyC not significantly increase in these in Fig. that GlyC is a nonabundant phospholipid in the whole cells and is formed during the dialysis, the cells are by osmotic shock. To study the course of the GlyC increase during the dialysis, we have analyzed by TLC lipids of equivalent cell aliquots at different times from the starting of dialysis and the of formed GlyC by video (Fig. the standard curves for the determination by video of the amounts of phospholipids and of PM are linear in the 1–10 range, to a quantitative analysis of the other major lipids present in the extracts before and after the dialysis, from 10 to 40 of lipid extracts were analyzed. By the cell lipid before and after the dialysis, it be that in with the GlyC increase, S-TGD-1 and data in Fig. the S-TGD-1 and decrease at the of dialysis in different At the of the dialysis, the ratio was found to be to that in isolated PM by NMR analyses (4Corcelli A. Lattanzio V.M.T. Mascolo G. Papadia P. Fanizzi F.P. Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane.J. Lipid Res. 2002; 43: 132-140Abstract Full Text Full Text PDF PubMed Google Scholar). Finally, video analysis that at the of dialysis, the minor PM lipid component BPG also (Fig. and content in high-producing BR H. salinarum cells before and after the dialysis. of lipid extracts of cells before and after dialysis have been on the The the of video analyses of S-TGD-1 and content before and after the dialysis in (1Lopez F. Lobasso S. Colella M. Agostiano A. Corcelli A. Light-dependent and biochemical properties of two different bands of bacteriorhodopsin isolated on phenyl-sepharose CL-4B.Photochem. Photobiol. 1999; 69: 599-604Crossref Scopus (25) Google A. Colella M. Mascolo G. Fanizzi F.P. Kates M. A novel glycolipid and phospholipid in the purple membrane.Biochemistry. 2000; 39: 3318-3326Crossref PubMed Scopus (73) Google M. Deroo P.W. Structure determination of the glycolipid sulfate from the extreme halophile Halobacterium cutirubrum.J. Lipid Res. 1973; 14: 438-445Abstract Full Text PDF PubMed Google A. Lattanzio V.M.T. Mascolo G. Papadia P. Fanizzi F.P. Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane.J. Lipid Res. 2002; 43: 132-140Abstract Full Text Full Text PDF PubMed Google B.F. Chang M. Duschl A. Lanyi J.K. Needlemann R. An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium.Gene. 1990; 90: 169-172Crossref PubMed Scopus (114) Google To light on the changes in lipid composition during the dialysis and to the that be the of some of lipids through the dialysis in we have the of the dialysis bag and studied the of osmotic on the lipid composition of H. salinarum by cells in different H. salinarum high-producing BR and were used in the following In to and the changes in GlyC and other membrane lipids in samples of and cells, lipids been extracted from equivalent aliquots of cells at different from the of the shock. During the osmotic shock, the total cell lipid content and the lipid/BR ratio not significantly the course of GlyC increase during the osmotic by video densitometry, together with the of osmotic fragility of high-producing BR halobacterial after the osmotic shock, 1 of GlyC over 40 of total lipids were formed in the cells (Fig. and the ratio was to not The lipid of the extracts of NRC-1 cell aliquots at different after in medium not only an increase in the GlyC content during the osmotic also a clear increase of BPG not The increase of BPG not be detected in high-producing BR cells of the very low content of h of cell incubation in of GlyC over of total lipid extract were formed in NRC-1 the ratio was found to be The of formed GlyC higher in NRC-1 cells than in high-producing BR cells under the same Finally, by the of extracts on the and the of we were to the conditions to by video analyses, that during the osmotic shock, in with GlyC increase, a decrease in S-TGD-1 content in NRC-1 cells to osmotic (Fig. The decrease in S-TGD-1 be detected only in the NRC-1 as in these cells the of formed GlyC is and the ratio is than in the high-producing BR halobacterial In addition, as previously found in the experiment in Fig. video analysis that also during the osmotic not is well as a phospholipid of the membrane of a of this phospholipid be a phospholipid The of cardiolipin in the of of as well as other been A. of the of phospholipids for 1973; Full Text PDF PubMed Google R. and properties of the of Full Text PDF PubMed Google of cardiolipin with of the its to the study of of PubMed Scopus Google Scholar). is also present in in to the of an for and have that a cardiolipin is located on the of the from of an cardiolipin and an membrane 1999; PubMed Scopus Google Scholar). In with containing only a of analogs of for in W. a novel from PubMed Scopus Google and in W. D. a major component of the lipid of PubMed Google Scholar). An or cardiolipin and a glycocardiolipin (GlyC) have been found residual lipids with delipidated BR fractions isolated from an engineered strain of H. salinarum (1Lopez F. Lobasso S. Colella M. Agostiano A. Corcelli A. Light-dependent and biochemical properties of two different bands of bacteriorhodopsin isolated on phenyl-sepharose CL-4B.Photochem. Photobiol. 1999; 69: 599-604Crossref Scopus (25) Google in the halophilic the of V.M.T. Corcelli A. Mascolo G. A. of two novel in the halophilic in the from the of and 2002; PubMed Scopus Google in a of from the of and in available from V.M.T. Corcelli A. Mascolo G. A. of two novel in the halophilic in the from the of and 2002; PubMed Scopus Google Scholar). GlyC is only present in BPG is in of for it in the halophilic for the of BPG with in an been A., S. P. A. and S. in isolated from an halophilic in Scholar); BPG be only a of PM. is the of in the of cardiolipin through the of other phospholipid while it is not the step a cardiolipin of or eukaryal data show for the first that the cardiolipin synthesis is by osmotic in the This study on the GlyC of the PM of H. GlyC is in PM with the whole cells, we previously suggested that GlyC is located only in the PM and not in other of the cellular of H. salinarum (2Corcelli A. Colella M. Mascolo G. Fanizzi F.P. Kates M. A novel glycolipid and phospholipid in the purple membrane.Biochemistry. 2000; 39: 3318-3326Crossref PubMed Scopus (73) Google Scholar). In this we have that the content of the cardiolipin GlyC in the extreme halophilic H. salinarum after osmotic shock, and that the formed GlyC is mainly accumulated in the PM. In other GlyC is in the PM of H. and its presence in isolated PM is induced by osmotic during the isolation The that GlyC is not present in the lipid extract of cells by French that the of cardiolipin during the dialysis is specifically due to osmotic and that it is not the of the cell of the used to the after cell not a increase of GlyC that salt not per increase GlyC and that the of the cell is to The of GlyC BPG) in cells osmotic before cell lysis and may the physiological of the to low the GlyC synthesis in H. salinarum a is not it be that GlyC be synthesized during PM isolation than by an also it is well that the of the PM is and other are present in PM. We have been to the of synthesized GlyC in H. salinarum cells by TLC video by the of various lipid components in the lipid extracts of and cells, we have found that in with the GlyC increase, a decrease of S-TGD-1 occurs. the glycolipid S-TGD-1 the same sugars in the same as GlyC in the polar it be suggested that the glycolipid S-TGD-1 is one of the for the last step of the of a of activated involved in the cardiolipin it to be its decrease during osmotic is with GlyC in this study show that the other minor cardiolipin BPG also in H. salinarum cells after osmotic we have not analyzed this in the of BPG by osmotic is more and to study in other of extreme halophiles a higher content of The presence of in an halophilic of the been described in a A., S. P. A. and S. in isolated from an halophilic in and we are the BPG in the presence of an osmotic shock. The increase of cardiolipin content in due to osmotic been in the A. H. in phospholipid composition of during of PubMed Scopus Google M. A. R. M. Lipid composition of and its PubMed Scopus Google Scholar). In it been that the de novo synthesis of cardiolipin during the of by osmotic A. H. in phospholipid composition of during of PubMed Scopus Google Scholar). been suggested that in the of cellular induced by osmotic the cardiolipin synthesis to increase the of the membrane M. A. R. M. Lipid composition of and its PubMed Scopus Google Scholar). The present study that a the osmotic fragility and the de novo synthesis of cardiolipin in halobacterial The for Mascolo for ESI-MS analyses, and for and PM This was by of and analog of bisphosphatidylglycerol, or cardiolipin bacteriorhodopsin electrospray ionization-mass spectrometry glycocardiolipin, or or S-TGD-1-PA phosphatidic acid purple membrane

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