Formation of acylphosphatidylglycerol by a lysosomal phosphatidylcholine:bis(monoacylglycero)phosphate acyl transferase

A delipidated soluble fraction prepared from a mitochondrial-lysosomal fraction of rabbit alveolar macrophages that catalyzes transacylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate was also found to transfer oleic acid from [14C]dioleoyl phosphatidylcholine to form acylphosphatidylglycerol. The reaction was dependent on the presence of bis(monoacylglycero)phosphate and was maximal at a concentration of 44 microM when the ratio of fatty acid transferred to fatty acid released was 0.28. Addition of phosphatidylglycerol had only a small effect. Homogenates of rat liver also catalyzed the reaction and after subcellular fractionation the activity was localized to lysosomes. The lysosomal activity was solubilized by delipidation with butanol to give a preparation with a specific activity 2462 times that of the homogenate. Optimal activity of soluble preparations from both macrophages and liver was at pH 4.5, with little activity above 6.0. Release of free fatty acid was also stimulated under conditions of optimal acyl transfer. Both acyl transfer and release of fatty acid were inhibited by Ca2+, detergents, chlorpromazine, lysophosphatidylcholine, and oleic acid. When there was disproportional inhibition, acyl transfer was always more affected. These results suggest that sequential acylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate and then acylphosphatidylglycerol constitute a mechanism in the lysosome for the transport and partition of fatty acids released by the lysosomal phospholipases.     

Products of endogenous N-acetylglucosaminyltransferase activity of Dictyostelium discoideum during growth and early development.

In the presence of Mn2+ and uridine diphosphate-N-acetyl-D-[14C]glucosamine, a total particulate fraction prepared from Dictyostelium discoideum amoebae catalyzed the transfer of N-acetyl[14C]glucosamine to endogenous membrane protein acceptors. No transfer to lipid acceptors was evident. The 14C products obtained from growth-phase and aggregation-competent amoebae were converted to glycopeptides by pronase digestion. The respective glycopeptides appeared identical in their chemical and chromatographic properties, suggesting that the same activity was functioning in both growing and differentiating cells. The results provided no evidence for developmental regulation of this activity in D. discoideum.     

Solubilization and properties of mannose and N-acetylglucosamine transferases involved in formation of polyprenyl-sugar intermediates.

A particulate fraction from porcine aorta catalyzed the incorporation of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc into both GlcNAc-pyrophosphorylpolyprenol and GlcNAc-GlcNAc-pyrophosphorylpolyprenol. This transfer utilized endogenous lipid and required a divalent cation. Mn2+ was the best metal ion and was optimum at 2.3 mM. This same particulate fraction was previously shown to transfer mannose from GDP-[14C]mannose to endogenous lipid to form mannosylphosphorylpolyprenol (Chambers, J., and Elbein, A.D. (1975) J. Biol. Chem. 250, 6904-6915). Both the GlcNAc activities and the mannose activity were solubilized by treatment of the particulate fraction with the detergent Nonidet P-40. The enzymes were partially purified by chromatography on DEAE-cellulose and on Sephadex G-200. These soluble enzymes required the addition of acceptor lipid for activity. An acidic lipid fraction, isolated from pig liver and having the properties of dolichyl phosphate, was active with either the GlcNAc or the mannose transferase. Chemically synthesized dolichyl phosphate was also active with either of these enzymes. The products formed from either GlcNAc or mannose by the soluble transferases were similar to those formed by the particulate enzyme. Thus the major product formed from UDP-[3H]GlcNAc was GlcNAc-pyrophosphoryldolichol with small amounts of the disaccharide-lipid while the product formed from GDP-[14C]mannose was mannosylphosphoryldolichol.     

Enzymatic synthesis of 2-O-alpha-D-mannopyranosyl-methyl-alpha-D-mannopyranoside by a cell-free particulate system of Mycobacterium smegmatis.

A cell-free particulate enzyme preparation of Mycobacterium smegmatis ATCC 607 catalyzed the transfer of labeled mannose from GDP[14C] mannose to methyl-alpha-D-mannopyranoside (an exogenously added acceptor) to form a product that was characterized to be 2-O-alpha-D[14C] mannopyranosyl-methyl-alpha-D-mannopyranoside. This transmannosylase activity was specific for both the sugar nucleotide donor and methyl monosaccharide acceptor. The reaction was stimulated by the addition of various metal ions and had a pH optimum of 6.0. The apparent Km of this transmannosylase reaction for methyl-alpha-D-mannopyranoside was 35 mM. The possible relationship between this "artificial" mannosyl-transfer system and the "natural" system which leads to the formation of the oligomannosides and glycoproteins is discussed.     

Glycolipid intermediates involved in the transfer of N-acetylglucosamine to endogenous proteins in a yeast membrane preparation.

A particulate membrane fraction from Saccharomyces cerevisiae contains transferases which catalyze the incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into a lipid fraction as well as into a protein fraction. The lipid fraction contains two alkali-stable lipids which can be separated on a silica G-60 column. The sugar moieties of these polyprenoid lipids are: N-acetylglucosamine and di-N-acetylchitobiose. The transfer of carbohydrate from isolated glycolipids to endogenous protein has been examined. After separation of protein and saccharide by hydrazinolysis and reacetylation only di-N-acetylchitobiose is found, and also when glycolipid containing only one N-acetylglucosamine is used as substrate. Maximum transfer of saccharides from glycolipids to protein is obtained at a Triton X-100 concentration of 1%. At this Triton X-100 concentration there is practically no transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the phosphorylated lipid. Therefore, when polyprenyl diphosphate N-acetyl[3H]-glucosamine is incubated together with UDP-N-acetyl[14C]glucosamine with the membrane fraction in the presence of 1% Triton X-100, a doubly labelled di-N-acetylchitobiose linked to lipid is formed with N-acetyl[14C]glucosamine at the non-reducing end of the chain.     

Biosynthesis of man-β-G1cNAc-G1cNAc-pyrophosphoryl-polyprenol by a solubilized enzyme from aorta

The particulate enzyme fraction from pig aorta was treated with Triton X-100 or Nonidet P-40 to yield a soluble enzyme preparation. This solubilized enzyme catalyzed the transfer of mannose from GDP-[¹⁴C]mannose, but not from [¹⁴C]mannosyl-phosphoryl-polyprenol, to G1cNAc-G1cNAc-pyrophosphoryl-polyprenol to form the trisaccharide-lipid, Man-β-GlcNAc-GlcNAc-pyrophosphoryl-polyprenol. The trisaccharide-lipid formed in these reactions was isolated by solvent fractionation and was subjected to mild acid hydrolysis to release the [¹⁴C]trisaccharide. Essentially all of the radioactivity was released from this trisaccharide as mannose upon treatment with β-mannosidase while α-mannosidase had no effect.     

Synthesis and secretion of lipids by long-term cultures of female rat hepatocytes.

The objective of this work was to characterize lipid metabolism in long-term cultures of adult rat hepatocytes from female rats and explore the potential use of this culture system to study the effect of hormones, drugs and toxic chemicals on it. Hepatocytes, seeded on a feeder layer of 3T3 cells, maintained for 2 weeks their typical morphology. The cultures were able to take up [14C]acetic and [14C]oleic acid from the culture medium and incorporate them into lipids. The synthesis and secretion of lipids by [14C]acetic acid-labeled cultures had a maximum value after 11 and 13 days in culture. Triacylglycerols were the main lipidic species synthesized and secreted by hepatocytes (up to 67% of the total lipids); they also synthesized and secreted phospholipids, cholesterol and cholesterol esters from [14C]acetic acid. Similarly, [14C]oleic acid-labeled cultures synthesized and secreted mostly triacylglycerols (up to 60-70% of the total lipids), but they were also able to incorporate the labeled precursor into both cellular and secreted phospholipids and cholesterol esters. The activity of glycerol-phosphate-dehydrogenase, marker enzyme of glycerolipid synthesis, decreased slightly during the culture time whereas the activity of malic enzyme, marker of fatty acid synthesis, increased. Our results show that long-term cultures of female rat hepatocytes are able to synthesize and secrete several lipids, specially triacylglycerols, from both [14C]acetic and [14C]oleic acid for at least 2 weeks and that they maintain enzyme activities related with the synthetic pathways of glycerolipids and fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of palmitate in perfused rat liver. Isolation of subcellular fractions containing triacylglycerol.

1. The metabolism of [1-14C]palmitate in rat liver was studied in a single-pass perfusion system at concentrations of 0.2 or 1 mM. 2. After the perfusion the liver was homogenized and the floating fat was isolated. The incorporation of [1-14C]palmitate into triacylglycerol in this pool increased 9-fold when the palmitate concentration in the medium was increased from 0.2 to 1 mM. In time studies with 1 mM-[1-14C]palmitate 75% of the total accumulation of triacylglycerol occurred in this pool. Our results support the concept that the floating-fat fraction contains the storage pool of triacylglycerol, i.e. the cytoplasmic lipid droplets. 3. In a particulate preparation consisting mainly of mitochondria and microsomal fraction the incorporation of [1-14C]palmitate into triacylglycerol was proportional to the fatty acid concentration. Triacylglycerol in the perfusate medium and in the particulate fraction was in isotopic equilibrium, which indicates that the particulate fraction contained the precursor pool for secreted triacylglycerol, i.e. the pool in endoplasmic reticulum and Golgi apparatus. 4. The oxidation to labelled water-soluble products and to CO2 was increased 14-fold by the 5-fold increase in palmitate concentration.     

Acylation of lysolecithin in the intestinal mucosa of rats

1. The presence of an active acyl-CoA-lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-(14)C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-(14)C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the beta-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-(14)C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order.     

Polyprenol phosphates and mannosyl transferases in Phaseolus aureus

The transfer of mannose from GDP[¹⁴C]mannose to lipid and to insoluble polymer by a particulate preparation of Phaseolus aureus has been investigated. The evidence favours the lipid being a prenol phosphate mannose. Of a range of prenol phosphates tried, betulaprenol phosphate was the most effective exogenous acceptor of mannose. Most of the insoluble [¹⁴C]polymer formed was glycoprotein in nature although small quantities of ¹⁴C were associated with glucomannan and galactoglucomannan fractions. Time studies failed to reveal a typical precursor-product relationship between the lipid and polymer fractions but on incubation of [¹⁴C]mannolipid with the particulate fraction a small transfer (0·5–0·7%) of [¹⁴C] to polymer was detected. p-Hydroxymercuribenzoate inhibited (by 90%) the transfer of [¹⁴C] from GDP[¹⁴C]-mannoseto polymer and simultaneously increased (3-fold) the [¹⁴C] recovered in the lipid fraction. The effect was nullified by mercaptoethanol. Attempts to solubilize the transfer system were only partially successful. The formation of a chromatographically identical mannolipid was demonstrated in particulate fractions of Codium fragile and tomato roots.     

A rapid effect of thyroxine on accumulation of diacylglycerols and on activation of protein kinase C in liver cells

L-Thyroxine rapidly stimulated the accumulation of diacylglycerols in isolated hepatocytes and in liver when lipids were prelabeled with [14C]oleic acid or with [14C]CH3COONa. Perfusion of the liver of hypothyroid animals with L-thyroxine-containing solution or incubation of liver fragments with the hormone increased the content of diacylglycerols in the liver cells. The increase in [14C]diacylglycerol level in the liver cells was accompanied by a decrease in the level of [14C]phosphatidylcholine, whereas contents of other 14C-labeled phospholipids, such as phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns4P), and phosphatidylinositol-4,5-bis-phosphate (PtdIns(4,5)P2), and of 14C-labeled fatty acids were the same as in the control. The L-thyroxine-induced accumulation of diacylglycerols in hepatocytes was not affected by neomycin but was inhibited by propranolol. Incubation of hepatocytes prelabeled with [14C]oleic acid with L-thyroxine and ethanol (300 mM) was accompanied by generation and accumulation of [14C]phosphatidylethanol that was partially suppressed by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). L-Thyroxine was responsible for the translocation of protein kinase C from the cytosol into the membrane fraction and for a many-fold activation of the membrane-bound enzyme. D-Thyroxine failed to affect the generation of diacylglycerols in hepatocytes and the activity of protein kinase C.     

Conversion of human placental alkaline phosphatase from a high Mr form to a low Mr form during butanol extraction. An investigation of the role of endogenous phosphoinositide-specific phospholipases.

Alkaline phosphatase in a wide range of tissues has been shown to be anchored in the membrane by a specific interaction with the polar head group of phosphatidylinositol. It has previously been suggested that the production of low Mr alkaline phosphatase during the commonly used butanol extraction procedure may result from the activation of an endogenous phosphoinositide-specific phospholipase C which removes the 1,2-diacylglycerol responsible for membrane anchoring. This conversion process was investigated in greater detail with human placenta used as the source of alkaline phosphatase. Mr and hydrophobicity of the alkaline phosphatase were determined by gel filtration on TSK-250 and partitioning in Triton X-114, respectively. Alkaline phosphatase extracted from human placental particulate fraction with butanol at pH 5.4 or released by incubation with Staphylococcus aureus phosphatidylinositol-specific phospholipase C produced a form of alkaline phosphatase of Mr approx. 170,000 and relatively low hydrophobicity. By contrast, the butanol extract prepared at pH 8.3 was an aggregated form of Mr approx. 600,000 and was relatively hydrophobic. The effect of a variety of inhibitors and activators on the amount of low Mr alkaline phosphatase produced during butanol extraction revealed that it was a Ca2+- and thiol-dependent process. Proteinase inhibitors had no effect. [3H]Phosphatidylinositol hydrolysis by the particulate fraction, unlike low Mr alkaline phosphatase production, was relatively sensitive to heat inactivation, indicating that the phosphoinositide-specific phospholipases C from cytosol and lysosomes were unlikely to be responsible for conversion. A butanol-stimulated activity which removed the [3H]myristic acid from the variant surface glycoprotein ( [3H]mfVSG) of Trypanosoma brucei was detectable in the human placental particulate fraction. Since this activity was acid active, Ca2+- and thiol-dependent and relatively heat stable, it may be the same as that responsible for production of low Mr alkaline phosphatase. The only 3H-labelled product identified was phosphatidic acid, suggesting that the [3H]mfVSG-cleaving activity is a phospholipase D. These data strongly support the proposal that production of low Mr alkaline phosphatase during butanol extraction is an autolytic process occurring as the result of an endogenous phospholipase. However, they also suggest that the lysosomal and cytosolic phosphoinositide-specific phospholipases C that have previously been described in many mammalian tissues are not responsible for this process.     

Synthesis of yeast wall glucan.

Saccharomyces cerevisiae was treated with a mixture of toluene and ethanol to make it permeable to small molecules. This treatment unmasked a glucan synthetase activity which was assayed with UDP-[U-14C]glucose. About 60% of the polymer formed was beta-(I leads to 3)glucan. No labelled lipids were detected. The 14C incorporated was recovered in a particulate membrane preparation isolated by differential centrifugation. When the particles themselves were assayed for glucosyl transfer activity none was found. The toluene-treated preparations also catalysed the transfer of mannosyl residues from GDP-mannose to polymeric materials by a process independent of glucosyl transfer.     

Biosynthesis of xyloglucan in suspension-cultured soybean cells. Occurrence and some properties of xyloglucan 4-beta-D-glucosyltransferase and 6-alpha-D-xylosyltransferase

A particulate enzyme fraction that catalyzes the transfer of glucose from UDP-[14C]glucose and of xylose from UDP-[14C]xylose into a xyloglucan has been isolated from suspension-cultured soybean cells. The incorporation of radioactivity from [14C]xylose into the polysaccharide was dependent on the presence of UDP-glucose in the incubation mixture, and that from [14C]glucose was dependent on the concentration of UDP-xylose in the mixture. Mn2+ was required for the incorporation of xylose and the optimum concentration of Mn2+ was about 10 mM. This reaction showed a pH optimum at 6.5 to 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and was inhibited by phosphate buffer and Tris buffer. On hydrolysis with Trichoderma endoglucanase, the polysaccharide synthesized in vitro gave a pentasaccharide, a hepatasaccharide, and a small amount of non-asaccharide. Based on the results from fragmentation and methylation analyses, the following structures were proposed for the penta- and the heptasaccharides from the xyloglucan synthesized in vitro: (formula, see text).     

Low-temperature-induced changes in intracellular fatty acid fluxes in Dunaliella salina

Two-minute exposures to exogenous [14C]palmitic, [14C]oleic, or [14C]lauric acid differentially labeled the lipids of Dunaliella salina microsomes and chloroplasts. Changes in fatty acid desaturation and intracellular movement during a subsequent 16-h incubation in nonradioactive medium indicated a slow transfer of lipids into the chloroplast from other organelles. Since Dunaliella lacks the massive traffic of microsomally produced glycerolipids into chloroplast galactolipids that dominates chloroplast-microsome lipid relations in most plant cells, it affords a sensitive system for studying more subtle intracellular lipid fluxes. Lowering the culture temperature from 30 to 12 degrees C was more inhibitory toward glycerolipid biosynthesis in chloroplasts than in microsomes. The ability of Dunaliella chloroplasts to utilize microsomal lipids may be essential for their systematic acclimation to low temperature.     

Phospholipase C from human sperm specific for phosphoinositides

Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization.     

Amphomycin inhibits the incorporation of mannose and GlcNAc into lipid-linked saccharides by aorta extracts

Amphomycin inhibits the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNac from UDP-[³H]GlcNAc into lipid-linked saccharides by either a particulate or a solubilized enzyme fraction from pig aorta. The solubilized enzyme was much more sensitive to the antibiotic than was the particulate fraction with 50% inhibition being observed at 8–15 μg of amphomycin. Although the antibiotic inhibited mannose transfer from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol, lipid-linked oligosaccharides and glycoprotein, the synthesis of mannosyl-phosphoryl-dolichol was much more sensitive to amphomycin. Amphomycin also inhibited the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryldecaprenol in particulate extracts of $\text{Mycobacterium smegmatis}$.     

Evidence for the presence of a glycosphingolipid-transfer protein in rat brain cytosol

Proteins in the postmicrosomal supernatant fraction of rat brain catalyzed the transfer of bovine brain galactocerebroside, sulfatide, and ganglioside GM1 from unilamellar liposomes to the rat erythrocytes or ghosts. The vesicles were made with egg yolk lecithin, cholesterol, 3H-labelled glycolipid, and a trace of [14C]triolein as a nonexchangeable marker. The routine assay of the glycosphingolipid transfer consisted of incubation of the donor liposomes with erythrocytes in the presence or absence of supernatant protein in physiological buffer at 37 degrees C for various time intervals. After the incubation, the erythrocytes were separated from the vesicles by centrifugation and the extent of protein-catalyzed transfer of labelled glycolipid in the membrane-bound total lipid fraction was determined by scintillation spectrometry. The fraction of [3H]glycosphingolipid transferred is represented by a change in the 3H/14C ratios at initial and subsequent time intervals. The glycosphingolipid transfer catalyzed by the supernatant protein was found to be logarithmic, whereas the protein-independent transfer was linear over a period of 3-4 h. The rate constant (K) and half time (t1/2) of the protein-catalyzed transfer reaction of cerebrosides and sulfatides were almost the same, while the transfer of ganglioside GM1 occurred at a slightly faster rate, probably owing to the greater aqueous solubility of this lipid. The transfer activity was also increased in a manner dependent on the amount of supernatant protein added up to 10 mg. The catalytic activity of the protein was lost when heated at 70 degrees C for 5 min. The pH optimum of the activity was around 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid biosynthesis and composition in oil bodies and microsomes of olive fruit

Both lipid synthesis and composition in oil bodies and microsomes of olive fruit at the first stage of development have been studied. The rate of fatty-acid synthesis in isolated oil bodies was saturated by 4.0 microM [2-14C]-malonyl-CoA. The fatty-acids synthesized of phospholipids and neutral lipids were saturated and monounsaturated. Neutral lipids, galactolipids and, above all, phospholipids were the major acyl-lipid components of microsomal fraction, oleic and palmitic being their principal fatty-acids. When the lipids of microsomes were labelled in vivo with [1-14C]-acetate, phospholipids and neutral lipids exhibited a higher biosynthesis rate relative to the galactolipids. The increase in saturated and monounsaturated fatty-acid synthesis in microsomes, was also accompanied by an important [1-14C]-acetate incorporation into polyunsaturated acids. The data presented here, in conjunction with our previous morphological results, suggest the possibility that olive fruit oil bodies could contain the necessary enzymes for the reserve lipid biosynthesis.     

Effects of low-temperature stress on the metabolism of phosphatidylglycerol molecular species in Dunaliella salina

A detailed analysis was made of individual phosphatidylglycerol (PG) molecular species isolated from microsomes and chloroplasts at various times after labeling Dunaliella salina cells with [14C]palmitic, [14C]oleic, or [14C]lauric acid. The patterns of [14C]fatty acid incorporation were in agreement with PG being formed by the "eucaryotic" type pathway in microsomes and the "procaryotic" type pathway in chloroplasts. In Dunaliella, which lacks a quantitatively significant flux of eucaryotic-type lipids from microsomes into chloroplast glycolipids, indications were found for a more subtle movement of microsomally synthesized PG into the chloroplasts. This transfer was more evident in cells stressed by exposure to 12 degrees C than it was at 30 degrees C, and may afford a mechanism for recruiting key microsomal PG molecular species toward low-temperature acclimation in chloroplasts.