The role of the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene in cytochrome oxidase assembly: mutation causes lowered levels of COX (cytochrome c oxidase) I and COX III mRNA

Abstract

Leigh syndrome French Canadian (LSFC) is a variant of cytochrome oxidase deficiency found in Québec and caused by mutations in the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene. Northern blots showed that the LRPPRC mRNA levels seen in skeletal muscle>heart>placenta>kidney>liver>lung=brain were proportionally almost opposite in strength to the severity of the enzymic cytochrome oxidase defect. The levels of COX (cytochrome c oxidase) I and COX III mRNA visible on Northern blots were reduced in LSFC patients due to the common (A354V, Ala354-->Val) founder mutation. The amount of LRPPRC protein found in both fibroblast and liver mitochondria from LSFC patients was consistently reduced to <30% of control levels. Import of [(35)S]methionine LRPPRC into rat liver mitochondria was slower for the mutant (A354V) protein. A titre of LRPPRC protein was also found in nuclear fractions that could not be easily accounted for by mitochondrial contamination. [35S]Methionine labelling of mitochondrial translation products showed that the translation of COX I, and perhaps COX III, was specifically reduced in the presence of the mutation. These results suggest that the gene product of LRPPRC, like PET 309p, has a role in the translation or stability of the mRNA for mitochondrially encoded COX subunits. A more diffuse distribution of LRPPRC in LSFC cells compared with controls was evident when viewed by immunofluorescence microscopy, with less LRPPRC present in peripheral mitochondria.

Figure 1. Northern blots to analyse transcript levels of LRPPRC and COX subunits in different tissues

Tissue-specific transcription analysis of LRPPRC by Northern blotting. The filter containing different tissue mRNAs was purchased from Clontech and hybridized with ³²P-labelled cDNA probes for LRPPRC, COX I, COX II, COX III and β-actin. The filter was exposed to an X-ray film to reveal the comparative labelling of the RNA species.

Figure 2. Northern blots to analyse the transcript level of LRPPRC and COX subunits in cultured skin fibroblasts

The same filter was probed in succession with ³²P-labelled cDNA probes for LRPPRC, COX I, COX II, COX III, ND1 and β-actin. Lane 1 is the control cell line 4212. Lanes 2, 3 and 4 are cell lines 7240, 8356 and 13912 from LSFC patients respectively. The experimental procedure is outlined in the Materials and methods section.

Figure 3. Western-blot analysis of LRPPRC contents of skin fibroblasts and liver mitochondria from LSFC patients and controls

The mitochondrial fraction was prepared by differential centrifugation and separated on an SDS/8% polyacrylamide gel. The separated samples were transferred on to a nitrocellulose membrane and blotted with antibodies to LRPPRC and the 49 kDa subunit of complex I (CI49k). After washing to remove unbound primary antibodies, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG and developed with ECL® solution. (A) LRPPRC content in fibroblast cell mitochondria. N, normal cells; H, heterozygous cells; LSFC, fibroblast cells from LSFC patients. (B) LRPPRC content of liver mitochondria. Lane 1, normal liver mitochondria; lanes 2–4, liver mitochondria from LSFC patients. Locations of LRPPRC and CI49K are indicated on the right side. Migration of standard proteins of designated molecular mass (MW) are marked on the left side. CI49K is the mitochondrial 49 kDa complex I protein, which was used as an equal loading indicator. (C) Import of [³⁵S]methionine-labelled LRPPRC into rat liver mitochondria. A full-length LRPPRC cDNA was translated in a reticulocyte lysate using [³⁵S]methionine incorporation as described in the Materials and methods section. Samples were taken for wild-type and A354V mutant LRPPRC incubated with rat liver mitochondria for 10, 20, 30 and 60 min and protein bands visualized after separation on an SDS/10% polyacrylamide gel. Bands are designated pre-LRPPRC and LRPPRC for the precursor and mature proteins respectively.

Figure 4. Western-blot analysis of LRPPRC content in nuclei

Nuclear fractions were prepared by removing cell membranes, organelles and cytoplasm in a low-salt 0.1% NP40 lysis buffer followed by extraction in a high-salt extraction buffer. Samples from either normal or LSFC fibroblast cells were separated on an SDS/10% polyacrylamide gel and transferred on to a nitrocellulose membrane. The membrane was probed with antibodies to LRPPRC, CI49K and histone H1 respectively. Upper panel: anti-LRPPRC and anti-CI49k blots. Lower panel: anti-histone H1 blot. Lane 1, normal mitochondrial fraction from cell line 4212; lanes 2 and 3, normal nuclear fraction from cell lines 4212 and 3839; lane 4, nuclear fraction from 4254 heterozygous cells; lanes 5–7, nuclear fraction from cell lines 3516, 7143 and 7240 from LSFC patients. CI49K is a mitochondrial-specific protein. Histone H1 is a nuclear-specific protein.

Figure 5. [³⁵S]Methionine pulse-labelled human mitochondrial translation products

Normal or patient fibroblast cells were pulse-labelled with [³⁵S]methionine in the presence of 100 μg/ml emetine for 90 min. The mitochondrial fraction was prepared by differential centrifugation. Radiolabelled protein was determined by scintillation counter. Total labelled protein (10000 c.p.m.) was loaded on each lane and separated on an SDS/polyacrylamide gel (10–20% linear gradient). The separated samples were transferred on to a nitrocellulose membrane by electrophoresis and the membrane was exposed to an X-ray film. MTCO1, MTCO2 and MTCO3 denote subunits 1, 2 and 3 of COX respectively. MTND1, MTND2, MTND3, MTND4, MTND4L, MTND5 and MTND6 refer to subunits of the rotenone-sensitive NADH dehydrogenase. ATP6 and ATP8 are subunits 6 and 8 of H⁺-ATPase. Migration of standard proteins is indicated on the left side. N, normal cells; H, heterozygous cells; LSFC, patient cells.

Figure 6. Immunostaining of LRPPRC in fibroblast cells

Both normal and LSFC fibroblast cells transfected with mitochondrially targeted red fluorescent protein were fixed, permeabilized and blocked under the same conditions. The treated cells were incubated with rabbit anti-LRPPRC antibody for 1 h and, after washing, incubated again with FITC-conjugated anti-rabbit antibody for a further 1 h. After removal of unbound secondary antibody, the cells were mounted in anti-fade plus DAPI solution. Fluorescence signals in the cells were visualized under the fluorescence microscope. Upper panel, fluorescence image in normal fibroblast cells; lower panel, fluorescence image in LSFC fibroblast cells. (i) Blue signal, DAPI stain of nucleus; (ii) mitochondrial red fluorescent protein; (iii) green signal, FITC-stained LRPPRC; and (iv) combined image to show overlap.