A mitochondrial phosphatase required for cardiolipin biosynthesis: the PGP phosphatase Gep4

Abstract

Cardiolipin (CL), a unique dimeric phosphoglycerolipid predominantly present in mitochondrial membranes, has pivotal functions for the cellular energy metabolism, mitochondrial dynamics and the initiation of apoptotic pathways. Perturbations in the mitochondrial CL metabolism cause cardiomyopathy in Barth syndrome. Here, we identify a novel phosphatase in the mitochondrial matrix space, Gep4, and demonstrate that it dephosphorylates phosphatidylglycerolphosphate to generate phosphatidylglycerol, an essential step during CL biosynthesis. Expression of a mitochondrially targeted variant of Escherichia coli phosphatase PgpA restores CL levels in Gep4-deficient cells, indicating functional conservation. A genetic epistasis analysis combined with the identification of intermediates of CL biosynthesis allowed us to integrate Gep4 in the CL-biosynthetic pathway and assign an essential function during early steps of CL synthesis to Tam41, which has previously been shown to be essential for the maintenance of normal CL levels. Our experiments provide the framework for the further dissection of mechanisms that are required for accumulation and maintenance of CL levels in mitochondria.

Figure 1

Δgep4 cells phenocopy CL-deficient cells. (A) Alignment of proteins homologous to Gep4. The region containing the inverted phosphatase motif DXDX(V/T) is shown. (B) Growth phenotypes of cells lacking functional Gep4. Serial dilutions of the indicated strains were spotted on YPD or synthetic growth (SC) media, which contained 1 M sorbitol or 25 μg/μl ethidium bromide (EtBr) when indicated, and incubated at 30 or 37°C. (C) Gep4 is required for the stability of respiratory chain supercomplexes. Mitochondria were isolated from cells indicated and solubilized with digitonin (4 g/g) and analysed by BN-PAGE and immunoblotting. Immunoblotting with myc-specific antibodies revealed equal expression of Gep4 and Gep4 variants thereof. III, complex III; IV, complex IV; V, F₁F_O-ATP synthase, mon, monomer, dim, dimer. (D) Synthetic lethal interaction of Δgep4 and Δups2. A diploid strain heterozygous for deletions of GEP4 and UPS2 was subjected to sporulation and tetrad dissection. Arrowheads indicate inviable double-mutant progeny.

Figure 2

Gep4 is a mitochondrial matrix protein attached to the inner membrane. (A) Subcellular fractionation. Whole-cell lysates (T) were centrifuged at 10 000 g to obtain a crude mitochondrial pellet and a postmitochondrial supernatant (PMS). The crude mitochondrial fraction was further centrifuged in a sucrose density gradient at 100 000 g for 1 h to remove ER membranes (Mito). The PMS was subjected to a clarifying spin, to remove residual mitochondrial membranes, at 30 000 g for 30 min and further centrifugation at 40 000 g for 30 min to obtain a microsomal membrane pellet (ER) (Gaigg et al, 1995). A measure of 40 μg of the cell lysate and the PMS and 20 μg of the ER and mitochondrial fraction were analysed by SDS–PAGE and immunoblotting with the indicated antisera. Sec61, Cox2 and Pgk1 served as marker proteins for ER, mitochondria and cytosol, respectively. The asterisk indicates an unspecific cross-reaction of the Gep4 antiserum. (B) Subfractionation of mitochondria. Mitochondria or mitoplasts generated by hypotonic disruption of the OM (+SW) were incubated with or without proteinase K (PK, 50 μg/ml) and analysed by SDS–PAGE and immunoblotting. The IMS protein Yme1 and the matrix protein F₁α served as controls. Mitochondrial membranes were solubilized with Triton X-100 (TX) (0.02%) when indicated. For control, mitochondria isolated from wild-type (WT) and Δgep4 cells were analysed in parallel. (C) Mitochondria (50 μg each sample) isolated from wild-type cells were treated with Na₂CO₃ (pH 11.5) or disrupted by sonication in the presence of 25, 100 or 250 mM NaCl. Extracts were separated into membrane (P) and soluble (S) fractions by ultracentrifugation (100 000 g, 30 min) and analysed by SDS–PAGE and immunoblotting. The integral inner membrane protein Cox2 and the soluble matrix protein Aco1 served as controls. T, input.

Figure 3

Gep4 is required for the maintenance of normal CL levels. (A) Mitochondria were isolated from cells indicated and the lipid composition was analysed by TLC (two top panels, the asterisk indicates an unidentified lipid species). Protein extracts were analysed by SDS–PAGE and immunoblotting using myc-specific antibodies and, as a loading control, Tom40 (two lower panels). (B) Mass spectrometric analysis of the phospholipid composition of mitochondria isolated from wild-type (WT), Δgep4 and Δcrd1 cells. Data represent mean values±s.d. of three (WT, Δgep4) or two (Δcrd1) independent mitochondrial isolations each analysed in duplicates. ^**P<0.005, ^***P<0.001. (C) Impaired CL synthesis in Δgep4 cells. WT, Δgep4 or Δcrd1 cells were grown in the presence of [³²P]_i for the indicated time periods. [³²P]_i incorporated into phospholipids was determined by TLC analysis and audioradiography (an unknown lipid species migrating at the same heights as CL is detected in all samples). PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; PA, phosphatidic acid; CL, cardiolipin.

Figure 4

Gep4 acts at the step of PGP dephosphorylation in CL biosynthesis. (A) Schematic representation of the CL biosynthesis. PA, phosphatidic acid; CDP-DAG, cytidine diphosphate-diacylglycerol; G3P, glycerol-3-phosphate; PGP, phosphatidylglycerolphosphate; PG, phosphatidylglycerol; DAG, diacylglycerol; FA, fatty acid; MLCL, monolysocardiolipin; MLPC, monolysophosphatidylcholine. (B) Epistasis analysis of the CL biosynthesis pathway. The mitochondrial phospholipid composition of the indicated cells was analysed by TLC (the asterisks indicate unidentified lipid species). (C) The E. coli PGP phosphatase PgpA can complement deficiencies of Δgep4 cells. Serial dilutions of indicated strains were spotted on YPD plates and incubated at 37°C (upper panel). Mitochondria were isolated from cells indicated and levels of CL, PE and PC were determined by TLC (middle panel, the asterisk indicates an unidentified lipid species). Extracts of Δgep4 and Δgep4+pYX142-Su9-PgpA mitochondria were analysed by SDS–PAGE and immunoblotting with antiserum specific for Gep4 (bottom panel).

Figure 5

PGP accumulates in Δgep4 mitochondria. (A) Mass spectrometric analysis of PGP and PG in wild-type, Δgep4 and Δcrd1 mitochondria. Data represent mean values±s.d. of three (WT, Δgep4) or two (Δcrd1) independent mitochondrial isolations each analysed in duplicates. PL, phospholipids. ^**P<0.005. (B) Detection of PGP in Δgep4 mitochondria by TLC. (C) Mass spectrometric profile of PGP. The lipid species are indicated by their fatty acid chain length and saturation state (e.g. PGP 34:1). The m/z values represent positively charged ammonium adducts of PGP. Selective scanning for PGP was done as described in Materials and methods. (D) Two-dimensional TLC of mitochondrial lipids from wild-type (WT) and Δgep4 cells. The identity of the spots was determined using synthetic phospholipid standards and, for control, mitochondrial phospholipids from Δtaz1 and Δcrd1 cells (the asterisks indicate unidentified lipid species).

Figure 6

Gep4 dephosphorylates PGP in vitro. (A) Gep4 and Gep4^D45N harbouring N-terminal hexahistidine peptides were expressed in E. coli and purified by Ni-NTA chromatography. Eluate fractions used for the in vitro experiments are shown. (B) In vitro PGP dephosphorylation assay. Purified Gep4 or Gep4^D45N were mixed with lipids extracted from Δgep4 mitochondria in assay buffer and incubated at 25°C for time periods indicated. The dephosphorylation of PGP (upper panel) and the formation of PG (bottom panel) were monitored (the asterisk indicates an unidentified lipid species). (C) TAM41 is epistatic to PGS1 and GEP4. Mitochondrial phospholipids isolated from the indicated strains were analysed by TLC. The bottom panel shows a section of the TLC in the upper panel using higher contrast settings (the asterisks indicate unidentified lipid species). (D) Growth of various CL-deficient strains on YPD or YPGal medium at 30°C.

Figure 7

The synthesis of CL in mitochondria. Gep4 and Tam41 are localized to the matrix space, peripherally attached to the inner membrane. Crd1 is an inner mitochondrial membrane protein catalytically active on the matrix side. Pgs1 lacks transmembrane regions and is predicted to be localized in the matrix. Cds1 is mainly present in the ER but Cds1 activity was also detected in mitochondrial inner membrane fractions. Whether Cds1 exerts its activity on the matrix side or the IMS side of the inner membrane remains unknown. Several lysophosphatidic acid acyltransferases (LPAATs) involved in PA biosynthesis are localized to the ER in yeast, the contribution of each of those to phospholipid biosynthesis remains to be determined. Whether PA is also provided by phospholipases D within or outside mitochondria remains unknown. CL is synthesized in the mitochondrial inner membrane but is also present in the mitochondrial outer membrane (Gebert et al, 2009). Therefore, mechanisms must exist that ensure transport from the inner to the outer membrane. Steps that might be affected by Tam41 are marked with a dashed circle. IM, inner mitochondrial membrane; OM, outer mitochondria membrane; MAM, mitochondria-associated membrane; LPAATs, lysophosphatidic acid acyltransferases.