LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum

<p>Abstract</p> <p>Background</p> <p>The phospholipids of the plant plasma membrane are synthesized in the endoplasmic reticulum (ER). The majority of these lipids reach the plasma membrane independently of the secretory vesicular pathway. Phospholipid delivery to the mitochondria and chloroplasts of plant cells also bypasses the secretory pathway and here it has been proposed that lysophospholipids are transported at contact sites between specific regions of the ER and the respective organelle, followed by lysophospholipid acylation in the target organelle. To test the hypothesis that a corresponding mechanism operates to transport phospholipids to the plasma membrane outside the secretory pathway, we investigated whether lysolipid acylation occurs also in the plant plasma membrane and whether this membrane, like the chloroplasts and mitochondria, is in close contact with the ER.</p> <p>Results</p> <p>The plant plasma membrane readily incorporated the acyl chain of acyl-CoA into phospholipids. Oleic acid was preferred over palmitic acid as substrate and acyl incorporation occurred predominantly into phosphatidylcholine (PC). Phospholipase A₂stimulated the reaction, as did exogenous lysoPC when administered in above critical micellar concentrations. AgNO₃was inhibitory. The lysophospholipid acylation reaction was higher in a membrane fraction that could be washed off the isolated plasma membranes after repeated freezing and thawing cycles in a medium with lowered pH. This fraction exhibited several ER-like characteristics. When plasma membranes isolated from transgenic <it>Arabidopsis </it>expressing green fluorescent protein in the ER lumen were observed by confocal microscopy, membranes of ER origin were associated with the isolated plasma membranes.</p> <p>Conclusion</p> <p>We conclude that a lysoPC acylation activity is associated with plant plasma membranes and cannot exclude a PC transacylase activity. It is highly plausible that the enzyme(s) resides in a fraction of the ER, closely associated with the plasma membrane, or in both. We suggest that this fraction might be the equivalent of the mitochondria associated membrane of ER origin that delivers phospholipids to the mitochondria, and to the recently isolated ER-derived membrane fraction that is in close contact with chloroplasts. The <it>in situ </it>function of the lysoPC acylation/PC transacylase activity is unknown, but involvement in lipid delivery from the ER to the plasma membrane is suggested.</p

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-3

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>ained 25 μg plasma membrane protein. , Distribution of radiolabel between 18:1-CoA (solid squares), free fatty acids (open squares) and phospholipids (open circles). , Incorporation of radiolabel into PC (filled symbols) and PE (open symbols). Either [C]18:1-CoA (solid line) or [C]16:0-CoA (dashed line) was used as substrate. The data represent mean values ± the range of duplicates from a representative experiment

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-10

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>eous polymer two phase counter current distribution of pea seedling microsome membranes (1–10; cf. Fig. 1), pea plasma membrane (PM) and pea microsome membranes (MS). The arrows mark the positions of molecular weight markers

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-4

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>centration of added lysophospholipid was 70 μM (lysoPC and lysoPE were predominantly acylated with 16:0 or 18:0, lysoPA was acylated with 18:1). Radiolabel recovery is presented for PC (solid bars), PE (cross-hatched bars) and PA (open bars). Otherwise as in Figure 4

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-6

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>fer containing 0.05% (w/v) Triton X-100 for 30 min, without or with the indicated additions (0.001 U bee venom PLA, 100 μM non-radiolabelled 18:1-CoA). , The distribution of radiolabel between lysoPC (cross-hatched bar), PC (solid bar) and free fatty acids (diagonally hatched bar). , The distribution of radiolabel between the -1 (diagonally hatched bar) and -2 (cross-hatched bar) positions of [C]PC. The data represent the mean values and range from duplicates

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-0

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>lting 10 membrane fractions analyzed. , The normalized activities of choline phosphotransferase for ER (solid triangles; 100 corresponds to 10 pmol·[mg protein]·min), cytochrome C oxidase for mitochondrial inner membrane (open triangles; 100 corresponds to 1.46 mmol·[mg protein]·min) and 1,3-β-glucan synthase for plasma membrane (solid squares; 100 corresponds to 0.53 μmol·[mg protein]·min). The dashed line shows the normalized protein distribution between the fractions. , The normalized chlorophyll content of thylakoid (open squares; 100 corresponds to 0.12 mg·[mg protein]), the normalized binding of a monoclonal anti-β-COP anti body for Golgi (solid circles; antibody binding only occurred to fractions 2–4, 100 corresponds to the highest binding), 1,3-β-glucan synthase for plasma membrane (solid squares; 100 corresponds to 0.43 μmol·[mg protein]·min). and the normalized distribution of protein between the fractions (dashed line). As all parameters could not be assayed on the fractions from a single 10-step membrane distribution, the panels A and B present the results from two independent experiments, with the plasma membrane marker and the protein distribution analyzed for both

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-9

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>lting 10 membrane fractions analyzed. , The normalized activities of choline phosphotransferase for ER (solid triangles; 100 corresponds to 10 pmol·[mg protein]·min), cytochrome C oxidase for mitochondrial inner membrane (open triangles; 100 corresponds to 1.46 mmol·[mg protein]·min) and 1,3-β-glucan synthase for plasma membrane (solid squares; 100 corresponds to 0.53 μmol·[mg protein]·min). The dashed line shows the normalized protein distribution between the fractions. , The normalized chlorophyll content of thylakoid (open squares; 100 corresponds to 0.12 mg·[mg protein]), the normalized binding of a monoclonal anti-β-COP anti body for Golgi (solid circles; antibody binding only occurred to fractions 2–4, 100 corresponds to the highest binding), 1,3-β-glucan synthase for plasma membrane (solid squares; 100 corresponds to 0.43 μmol·[mg protein]·min). and the normalized distribution of protein between the fractions (dashed line). As all parameters could not be assayed on the fractions from a single 10-step membrane distribution, the panels A and B present the results from two independent experiments, with the plasma membrane marker and the protein distribution analyzed for both

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-2

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>lting membrane fractions were incubated with [C]18:1-CoA to monitor acyl incorporation into phospholipids. Filled circles, acyl incorporation into PC; open circles, acyl incorporation into PE; filled diamonds, the PE/PC ratio of acyl incorporation

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum-1

<p><b>Copyright information:</b></p><p>Taken from "LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum"</p><p>http://www.biomedcentral.com/1471-2229/7/64</p><p>BMC Plant Biology 2007;7():64-64.</p><p>Published online 28 Nov 2007</p><p>PMCID:PMC2241621.</p><p></p>ueous polymer two phase counter current distribution of pea seedling microsome membranes (1–10; cf. Fig. 1), pea plasma membrane (PM) and pea microsome membranes (MS). The arrows mark the positions of molecular weight markers