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. 2004 Sep 1;382(Pt 2):527-33.
doi: 10.1042/BJ20031819.

Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria?

Clara Bionda  1 , Jacques Portoukalian, Daniel Schmitt, Claire Rodriguez-Lafrasse, Dominique Ardail
Affiliations

Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria?

Clara Bionda et al. Biochem J. .

Abstract

Recent studies by our group and others have disclosed the presence of ceramides in mitochondria, and the activities of ceramide synthase and reverse ceramidase in mitochondria have also been reported. Since a possible contamination with the ER (endoplasmic reticulum)-related compartment MAM (mitochondria-associated membrane) could not be ruled out in previous studies, we have re-investigated the presence of the enzymes of ceramide metabolism in mitochondria and MAM highly purified from rat liver. In the present paper, we show that purified mitochondria as well as MAM are indeed able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabelled sphingosine as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward FB1 (fumonisin B1) in mitochondria as well as in MAM was found to depend upon the sphingoid base: whereas dihydrosphingosine N-acyltransferase was inhibited by FB1 in a concentration-dependent manner, FB1 actually activated the ceramide synthase when using sphingosine as a substrate. Acylation of sphingosine 1-phosphate and dihydrosphingosine 1-phosphate, generating ceramide 1-phosphate, was also shown with both subcellular fractions. Moreover, the same difference in sensitivity towards FB1 for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases. Collectively, these results demonstrate the involvement of mitochondria in the metabolism of ceramides through different pathways, thereby supporting the hypothesis that topology of ceramide formation could determine its function.

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Figures

Figure 1
Figure 1. Ceramide biosynthesis by purified mitochondria and MAM in vitro
Purified mitochondria and MAM were incubated with [3H]sphingosine and unlabelled palmitoyl-CoA under the conditions described in the Experimental section. After purification of the total lipid extract on solid-phase LC-NH2 columns, the fractions corresponding to ceramides (fractions 2 according to [28]) were analysed on silica gel TLC in the solvent system chloroform/methanol (12.5:1, v/v), along with standards. (A) Standards visualized at 150 °C after spraying with copper acetate (3%) in phosphoric acid (8%). Lane 1, ceramides type III; lane 2, phytoceramides. (B) Autoradiogram of fractions 2 eluted from LC-NH2 columns. Lane 1, mitochondria; lane 2, MAM. Arrows indicate the spots that were scraped and re-run in Figure 2.
Figure 2
Figure 2. TLC of the radiolabelled products obtained through ceramide synthase activity
Upper panel: the spots co-migrating in Figure 1 respectively as standard ceramide type III and phytoceramide were scraped from the plate and re-run on an HPTLC plate in chloroform/methanol (12.5:1, v/v). The plate was then cut into 0.5 cm portions, extracted with chloroform/methanol (2:1, v/v) and counted for radioactivity after evaporation of the organic solvents. X, phytoceramide; Y, ceramide. Lower panel: the radioactive spots from the upper panel were scraped from the plate, extracted with chloroform/methanol (2:1, v/v) and re-run in chloroform/methanol (19:1, v/v) on a silica gel plate pre-coated with 1% sodium meta-arsenite. The plate was scraped into 0.5 cm portions, extracted with chloroform/methanol (2:1, v/v) and the radioactivity was counted after evaporation of the organic solvents. X, phytoceramide; Y, ceramide.
Figure 3
Figure 3. Time course of conversion of radioactive [3H]sphingosine into ceramide
Incubation conditions were as described in the Experimental section, except that time was varied as indicated. Radioactive ceramides were purified as in Figure 1 and were separated on TLC, before scraping and liquid-scintillation counting. Inset, increasing protein concentrations were used for 5 min incubations. Results are means±S.D. for three distinct experiments.
Figure 4
Figure 4. Effect of FB1 on mitochondrial ceramide synthase in vitro
Purified mitochondria were pre-incubated for 10 min at 37 °C in the presence of 20 μM FB1, after which ceramide synthase assays were performed as described in the Experimental section with [3H]sphingosine or [3H]dihydrosphingosine as substrates. After a 15 min incubation time, total lipids were fractionated on LC-NH2 columns and analysed by silica gel TLC and autoradiography. Radioactive ceramides co-migrating with authentic standards were scraped and radioactivity was estimated by liquid-scintillation counting. Results are means±S.D. for four distinct experiments. (A) Dihydrosphingosine N-acyltransferase. (B) Sphingosine N-acyltransferase.
Figure 5
Figure 5. Substrate specificity of ceramide synthase in MAM and submitochondrial compartments in vitro
Purified subcellular fractions (100 μg of protein) were incubated for 15 min at 37 °C with 10 μM sphingosine or phytosphingosine and 2 nmol of [14C]palmitoyl-CoA. After purification of the total lipid extract on solid-phase LC-NH2 columns, fractions corresponding to ceramides were submitted to mild alkaline hydrolysis and analysed by silica gel TLC and autoradiography. Radioactive components co-migrating with authentic ceramides were scraped from the plate and radioactivity was estimated by liquid-scintillation counting. Results are means±S.D. for three distinct experiments. (A) MAM. (B) Purified mitochondria. (C) Purified outer mitochondrial membranes. (D) Purified inner mitochondrial membranes.
Figure 6
Figure 6. Comparative acylation of exogenous phosphorylated sphingoid bases in mitochondria (A) and MAM (B)
Subcellular fractions (100 μg of protein) were incubated for 30 min at 37 °C in the presence of either 10 μM sphingosine 1-phosphate or dihydrosphingosine 1-phosphate and 2 nmol of [14C]palmitoyl-CoA (with or without 20 μM FB1). The radioactive lipids were submitted to mild alkaline hydrolysis and purified further on LC-NH2 columns. Ceramides (eluted in fraction 2) and ceramide 1-phosphate or dihydroceramide 1-phosphate (eluted in fraction 6) was applied on TLC and run in chloroform/methanol (12.5:1, v/v) for ceramides or chloroform/methanol/ethanoic acid (6.5:1.5/1, by vol.) for ceramide 1-phosphate. After autoradiography, radioactive spots co-migrating with authentic standards were scraped from the plate and radioactivity was estimated by liquid-scintillation counting. Results are the means±S.D. for three distinct experiments. Cer, ceramide.
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