Phospholipid scramblase-3 regulates cardiolipin de novo biosynthesis and its resynthesis in growing HeLa cells

Abstract

PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell 'senses' altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.

Figure 1. Incorporation of [1,3-³H]glycerol into CL and PG in control, PLS3 and F258V cells

Vector (control) HeLa cells, HeLa cells overexpressing PLS3 (PLS3) or HeLa cells expressing the PLS3 mutant (F258V) were incubated for 8 h with [1,3-³H]glycerol and the radioactivity incorporated into CL (A) and PG (B) was determined as described in the Materials and methods section. Values represent the mean±S.D. of three experiments. *P<0.05.

Figure 2. Expression of hCLS1 mRNA in control, PLS3 and F258V cells

Total mRNA was isolated from HeLa cells (control), HeLa cells overexpressing PLS3 (PLS3) or HeLa cells expressing the PLS3 mutant (F258V) and the relative levels of CLS mRNA determined as described in the Materials and methods section. (A) Lane 1, control; lane 2, PLS, lane 3, F258V. Top panel, hCLS1; bottom panel, β-actin. A representative gel is presented. (B) Ratio of the relative intensity of hCLS1 to β-actin. The relative intensities of the bands were analysed by scanning the film, and were subsequently determined by Scion Image software. Values represent the means±S.D. of three experiments. *P<0.05.

Figure 3. [1-¹⁴C]linoleic acid incorporation into CL in control, PLS3 and F258V cells

HeLa cells (control), HeLa cells overexpressing PLS3 (PLS3) or HeLa cells expressing PLS3 mutant (F258V) were incubated with 0.1 mM [1-¹⁴C]linoleic acid for 8 h and the radioactivity incorporated into CL determined as described in the Materials and methods section. Values (expressed as dpm/mg protein) represent the means±S.D. of three separate experiments. *P<0.05.

Figure 4. Western blot of mitochondrial MLCL AT and expression of ALCAT1 mRNA in control, PLS3 and F258V cells

(A) Cellular protein fractions were prepared and Western blot analysis of mitochondrial MLCL AT in control HeLa cells (lane 1), F258V cells (lane 2) or PLS3 cells (lane 3) was performed using an anti-MLCL AT antibody and an anti-β-actin antibody as described in the Materials and methods section. A representative gel is presented. The relative intensities of the bands were analysed by scanning the film and are shown on the right. (B) Total mRNA was isolated from control HeLa cells (lane 1), F258V cells (lane 2) or PLS3 cells (lane 3) and the relative levels of ALCAT1 mRNA determined as described in the Materials and methods section. A representative gel is presented. The relative intensities of the bands were analysed by scanning the film and are depicted on the right. Values (expressed as dpm/mg protein) represent the means±S.D. of three separate experiments. *P<0.05.