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On the Export of Fatty Acids from the Chloroplast

2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês

10.1074/jbc.m311305200

1083-351X

Abraham J. Koo, John B. Ohlrogge, Mike Pollard,

Microbial Metabolic Engineering and Bioproduction

The model for export of fatty acids from plastids proposes that the acyl-ACP (acyl carrier protein) product of de novo fatty acid synthesis is hydrolyzed in the stroma by acyl-ACP thioesterases and the free fatty acid (FFA) released is then transferred to the outer envelope of the plastid where it is reactivated to acyl-CoA for utilization in cytosolic glycerolipid synthesis. Experiments were performed to assess whether the delivery of nascent FFA from the stroma for long chain acyl-CoA synthesis (LACS) occurs via simple diffusion or a more complex mechanism. The flux through the in vivo FFA pool was estimated using kinetic labeling experiments with spinach and pea leaves. The maximum half-life for FFA in the export pool was ≤1 s. Isolated pea chloroplasts incubated in the light with [14C]acetate gave a linear accumulation of FFA. When CoASH and ATP were present there was also a linear accumulation of acyl-CoA thioesters (plus derived polar lipids), with no measurable lag phase (<30 s), indicating that the FFA pool supplying LACS rapidly reached steady state. The LACS reaction was also measured independently in the dark after in situ generated FFA had accumulated yielding estimates of LACS substrate-velocity relationships. Based on these experiments the LACS reaction with in situ generated FFA as substrate is only about 3% of the LACS activity required in vivo at the very low concentrations of the FFA export pool calculated from the in vivo experiment. Furthermore, bovine serum albumin rapidly removed in situ generated FFA from chloroplasts, but could not compete effectively for "nascent" FFA substrates of LACS. Together the data suggest a locally channeled pool of exported FFA that is closely linked to LACS. The model for export of fatty acids from plastids proposes that the acyl-ACP (acyl carrier protein) product of de novo fatty acid synthesis is hydrolyzed in the stroma by acyl-ACP thioesterases and the free fatty acid (FFA) released is then transferred to the outer envelope of the plastid where it is reactivated to acyl-CoA for utilization in cytosolic glycerolipid synthesis. Experiments were performed to assess whether the delivery of nascent FFA from the stroma for long chain acyl-CoA synthesis (LACS) occurs via simple diffusion or a more complex mechanism. The flux through the in vivo FFA pool was estimated using kinetic labeling experiments with spinach and pea leaves. The maximum half-life for FFA in the export pool was ≤1 s. Isolated pea chloroplasts incubated in the light with [14C]acetate gave a linear accumulation of FFA. When CoASH and ATP were present there was also a linear accumulation of acyl-CoA thioesters (plus derived polar lipids), with no measurable lag phase ( 85% recovery of oleoyl-CoA either in the presence or absence of BSA. The Total in Vivo Pool of FFA in Leaves Is Very Small and Implies a Very Short Half-life for the FFA Pool Involved in Export from the Chloroplast-Although it is generally known that labeled FFA are minor products when leaf tissue is incubated with acetate, the amount of FFA has not previously been quantified. We have shown that FFA are intermediates in fatty acyl group export from the chloroplast (12Pollard M. Ohlrogge J. Plant Physiol. 1999; 121: 1217-1226Crossref PubMed Scopus (66) Google Scholar) and to better understand this export process it was important to quantify this FFA pool. Fig. 2 presents a time course for acetate incorporation into total fatty acids and into FFA by pea leaves over a 20 min period. With expanding pea leaves a linear rate of lipid synthesis was established within 2 min (Fig. 2A). At all time points the labeled total lipid extract from pea leaves contains 80-85% labeled fatty acids, mainly as phosphatidylcholine. Presumably the short lag phase indicates the time to reach the steady state balance between transport processes and pool filling in the biosynthetic utilization of acetate. To measure the FFA pool the leaf assays were quenched while still in the presence of substrate in the light (Fig. 2B). Control extractions spiking unlabeled tissue with [14C]oleic acid gave >90% recovery of label. In the experiment in Fig. 2 the labeled FFA pool at 2 min was 4.3% of total labeled lipids and contained essentially only palmitic and oleic acids. In pea most of the fatty acids synthesized in the chloroplast are exported (∼90%). The turnover time of FFA during plastid export for pea can be estimated as follows. Let the steady state rate of lipid synthesis be 100 units per min, of which ∼85 units are fatty acids. Therefore 76.5 units (90% of 85%) must pass through the FFA transfer pool for export to the eukaryotic pathway. At the 2 min time point the steady state level of labeled FFA in the transfer pool has been reached, and because the steady state rate extrapolates back to the x-axis at 1 min (Fig. 2A) the total amount of lipids synthesized is equivalent to about 100 units. Thus 100 units × 4.3% of labeled FFA are present (4.3 units). At 2 min we have a flux of 76.5 units per min through a transfer pool of 4.3 units. This gives a turnover time of 60 × 4.3/76.5 = 3.4 s and a t½ value of 2.35 s (t½= turnover time × 0.693). A repeat of the time course with pea leaves gave the onset of steady state labeling within 1 min, while the labeled FFA pool at 2 min was 2.05 ± 0.26% (4 determinations), giving a t½ value of 1.7 s. A similar time course and FFA pool analysis experiment was performed with spinach leaf strips (data not shown). A turnover time 0.96 s and a t½ value of 0.66 s were determined. These calculations for pea and spinach leaves represent estimated values for the FFA transfer pool turnover times that are an upper limit. The observed FFA pool may include the transient pool of FFA being exported from the plastid, FFA released from labeled acyl thioester pools during the heat quench step, plus FFA derived from other sources. The kinetics of labeled FFA appearance in the time course for pea leaves (Fig. 2B) give us an indication that much of the labeled FFA pool is not a transfer pool. If the observed FFA pool were all the transfer pool then this pool would saturate by 2 min. Instead the pool continues to grow substantially. We speculate that some of the FFA pool actually derives from fatty acid synthesis at the damaged margins of the leaf strips in the assay. Given the approximations in the calculations it seems reasonable to state that the t½ for FFA in any chloroplast export pool is about 1 s, or less, and possibly much less. Having established this limiting in vivo pool size we then examined the detailed relationship between products of fatty acid synthesis in isolated, intact pea chloroplasts. In pea, compared with spinach, much more of the fatty acid produced is exported from the chloroplast. Time Course for Acetate Incorporation into Fatty Acyl Products by Isolated Pea Chloroplasts-The synthesis of fatty acids from acetate by intact, illuminated chloroplasts can produce a variety of acyl products (34Heinz E. Roughan P.G. Plant Physiol. 1983; 72: 273-279Crossref PubMed Google Scholar, 35Gardiner S.E. Heinz E. Roughan P.G. Plant Physiol. 1984; 74: 890-896Crossref PubMed Google Scholar), depending on the plant species from which the chloroplasts are isolated and on the cofactors in the chloroplast incubation medium. In a minimal chloroplast incubation medium (no CoASH, glycerol-3-phosphate, nor UDP-galactose) the major product is free fatty acid (FFA). Smaller amounts of 1,2-diacylglycerol (DAG) (∼5-20%) and polar lipids (PL) (∼10-20%) have also been observed. When CoASH and ATP are added, up to 60% of the label may be found as acyl-CoA (11Roughan P.G. Slack C.R. FEBS Lett. 1981; 135: 182-186Crossref Scopus (10) Google Scholar). The incorporation of acetate into acyl lipids by pea chloroplasts is shown in Fig. 3. In minimal chloroplast incubation medium the [14C]acetate incorporation into total fatty acids was 15.5 pmol of fatty acid/s/mg chlorophyll (Fig. 3A), but when CoASH and ATP were present this rate increased 1.4-fold to 21.5 pmol of fatty acid/s/mg chlorophyll (Fig. 3B). In the minimal medium the major product was FFA (about 90% of total label). When CoASH and ATP were present up to 80% of the label was found as acyl-CoA plus polar lipids (PL). Fig. 3B shows that over a 35 min period in the light with ATP and CoASH total fatty acid synthesis was linear, and that the appearance of the individual products, namely FFA (∼25%), acyl-CoA (∼40%), and total PL (∼40%) were also linear, with negligible lag phase detected. The labeled PL fraction was composed of about 60% phosphatidylcholine and 10% phosphatidylglycerol. To confirm the lack of a lag phase earlier time points were taken (Fig. 3C). Any lag phase required for the establishment of the steady state condition of product accumulation in the presence of CoASH plus ATP was less than 30 s. In Fig. 3, B or C if the bulk FFA pool were a precursor of acyl-CoA, which is subsequently used for PL synthesis, a measurable lag would occur in acyl-CoA plus PL accumulation. Thus there was no observable precursor-product kinetic relationship between labeled FFA and acyl-CoA. This implies that the FFA concentration to run the LACS reaction at steady state rate has been reached rapidly ( 10 s by the gentle mixing required for delicate organelles like chloroplasts, for pool filling from acetate through to end products of fatty acid synthesis, and quench times. The Long Chain Acyl-CoA Synthesis (LACS) Reaction in the Dark Utilizing in Situ Generated FFA-When FFA is exported from the plastid it is converted to acyl-CoA for utilization in cytosolic reactions of lipid biosynthesis. The LACS reaction has previously been studied after preparation of chloroplast envelope vesicles and addition of FFA substrate (7Joyard J. Stumpf P.K. Plant Physiol. 1981; 67: 250-256Crossref PubMed Google Scholar). In this study we investigated the LACS reaction in intact pea chloroplasts with in situ generated FFA. Fig. 4 presents the effect of switching off the light after 15 min of incubation of chloroplasts in media containing no CoASH during the light period but added at the onset of the dark period, or in media containing CoASH during both light and dark periods. As observed by many researchers (33Browse J. Roughan P.G. Slack C.R. Biochem. J. 1981; 196: 347-354Crossref PubMed Scopus (87) Google Scholar, 36Roughan P.G. Kagawa T. Beevers H. Plant Sci. Lett. 1980; 18: 221-228Crossref Scopus (21) Google Scholar) total fatty acid synthesis was completely halted in the dark. What is noteworthy is the very different behavior of the individual acyl pools during the dark period. In the absence of CoASH the major fatty acid product in the light was FFA (Fig. 4A). When CoASH (0.5 mm) was added at the transition to darkness there was a very rapid conversion of FFA to acyl-CoA and its metabolites (PL). The initial rate of FFA depletion (from 15 to 20 min) was at least twice the rate of total fatty acid synthesis in the light. By contrast, in Fig. 4B the initial rate of FFA depletion on transition to darkness was less than 30% that of the rate of fatty acid synthesis in the light. The combined acyl-CoA and PL accumulation had essentially stopped in the dark period. Because the LACS reaction with in situ generated FFA was very fast in Fig. 4A this experiment was repeated with additional measurements immediately after the light to dark transition (Fig. 4C). There were variations between five independent experiments but upon the dark transition with coincident addition of CoASH, about 25-50%, and sometimes >50% of the FFA label was removed by the LACS reaction in the first 30 s. Thus the LACS reaction in chloroplasts has the capacity to run at a rate over 10 times that of fatty acid synthesis. However, the LACS reaction did not rapidly go to completion. Inspection of Fig. 4C shows that disappearance of FFA does not follow simple first order kinetics. There are at least two components to the disappearance of FFA; a rapid rate of depletion accounting for at least 60-70% of the labeled FFA but sometimes up to 80-85% depending on experiment, and a slow decay for the remainder. Evidence for Distinct Kinetic Pools of FFA in Chloroplast Assays-Based on Fig. 4A we know the LACS reaction is highly active in the dark and therefore the slow rate of LACS in Fig. 4B appears somewhat paradoxical, especially since in Fig. 4A the amount of labeled FFA at the dark transition is 4.7 nmol/mg chlorophyll while in Fig. 4B it is still 3.