Mutations in LYRM4, encoding iron-sulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes

Abstract

Iron-sulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called iron-sulfur or Fe-S proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203G>T, p.R68L) was identified via massively parallel sequencing of >1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other Fe-S proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of Fe-S proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.

Figure 1.

Pedigree of the extended families of patients P1 and P2. P1 and P2 were double first cousins from consanguineous Lebanese/Syrian families. There were at least two consanguineous loops within their parents' sibships with the same degree of consanguinity. Numbers within symbols describe the number of siblings of that gender. The proband is indicated by an arrowhead.

Figure 2.

Homozygous LYRM4 mutation identified in two patients with combined complexes I, II and III OXPHOS deficiency, predicted to affect a highly conserved amino acid residue. (A) Chromatograms of Sanger sequencing of gDNA confirmed the homozygous c.203G>T (p.R68L) mutation detected by MitoExome sequencing in P1 and identified the same mutation in P2. This mutation is within exon 2, which is one of the shared exons among all three LYRM4 transcript variants. (B) Arg68 in human ISD11 (NP_065141.3) is highly conserved in vertebrate and invertebrate species shown in the ClustalW2 alignment.

Figure 3.

SNP array profiles (Illumina KaryoStudio software) for chromosome 6 of P1 and P2. Frequencies of the B alleles along the chromosomes are indicated, where A and B are arbitrary labels for two alternative alleles. The ‘logR’ values reflect copy numbers, where 0.00 suggests copy number is equal to 2, whereas <0 and >0, respectively, suggest loss and gain of copy number. LYRM4 is located within the region of LCSH shared between P1 and P2 on chromosome 6. The deviations in ‘logR’ values for P2 were genomic waves due to the sub-optimal DNA quality and did not reflect genuine copy number change.

Figure 4.

SDS–PAGE western blot panel of patients' muscle and liver samples. In P2 muscle, ISD11 protein was undetectable and protein levels of all complex I–IV subunits investigated were either severely reduced or too low to be detected. In both patient liver samples (P1 and P2), ISD11 protein was undetectable and SDHB and UQCRFS1 levels were most severely reduced. COX1 and COX2 were only reduced in P2 but not in P1 liver. The aconitases and ferrochelatase in patient muscle and liver samples were either not detectable or reduced. C1 was used as muscle control, C2 and C3 as liver controls. P1 muscle sample was not available for analysis. At least one subunit from each OXPHOS complex was investigated: NDUFB8 and NDUFS3 from complex I (CI), SDHA and SDHB from complex II (CII), UQCRC2 and UQCRFS1 from complex III (CIII), COX1 and COX2 from complex IV (CIV), and ATP5A1 from complex V (CV). SDHB, UQCRFS1, aconitase 1 (cytosolic) and 2 (mitochondrial), and ferrochelatase are Fe–S proteins. ATP5A1 and porin were loading controls. Non-specific bands were marked with asterisks. Quantification of proteins was done by densitometry and results are shown in Supplementary Material, Table S1.

Figure 5.

Fitness of the pISD11R71L yeast strain in competitive growth assays. Each bar is the average and standard deviation of three independently generated clones grown in the given media in quadruplicate. All strains were grown in competition with a wild-type strain where ISD11 had not been mutated. The two test strains were deleted for ISD11 and the deletion covered with the given plasmids (deletion and plasmid were generated in a diploid to avoid selection for supressors). Competitive growth was performed in four media: synthetic minimal 2% dextrose (SC), synthetic minimal 2% galactose (SGal), synthetic minimal 2% acetate (SAce) and YPD. Growth differences were measured for 13 generations.

Figure 6.

Size exclusion chromatography of co-purified NFS1Δ1-55/ISD11 and NFS1Δ1-55/ISD11-R68L complexes. Complexes containing either NFS1Δ1-55/ISD11 or NFS1Δ1-55/ISD11-R68L eluted at a molecular mass of ∼145 kDa, corresponding to a complex with an NFS1 dimer with a stoichiometry of ISD11 subunits of 1:1 or 1:2. Another eluted peak corresponds to an NFS1 monomer, while the left-most peak corresponds to an aggregate product of the NFS1/ISD11 complex. NFS1/ISD11 labeling refers to complexes of NFS1Δ1-55 with either ISD11 or ISD11-R68L. Inset: Plot of the standard proteins.

Figure 7.

Influence of ISCU and FXN on the l-cysteine desulfurase activity of NFS1Δ1-55/ISD11 and NFS1Δ1-55/ISD11-R68L complexes, and NFS1Δ1-55. l-Cysteine desulfurase activities of NFS1Δ1-55/ISD11 (black), NFS1Δ1-55/ISD11-R68L (white) or NFS1Δ1-55 (grey) were determined in the presence of 1 mm l-cysteine, with or without the addition of ISCU or/and FXN. Desulfurase activity of the NFS1Δ1-55/ISD11 complex (black) was unaffected by FXN alone, decreased 3-fold with ISCU alone, but doubled when both ISCU and FXN were present. Desulfurase activity of the NFS1Δ1-55/ISD11-R68L complex (white) was grossly decreased compared with wild-type complex, and increased when incubated with both ISCU and FXN, but remained deficient compared with the wild-type complex. Purified NFS1Δ1-55 was almost inactive without ISD11 and only attained a detectable residual activity when ISCU and FXN were added to the assay. Data are represented as mean ± standard deviations determined from at least three independent experiments.

Figure 8.

Growth curves of E. coli CL100(ΔiscS)(DE3) strain transformed with NFS1Δ1-55/ISD11, NFS1Δ1-55/ISD11-R68L and IscS expression plasmids. E. coli lacking IscS exhibit a striking growth defect (negative control), which is able to be reversed with the expression of a plasmid containing IscS (E.coli IscS). The growth defect was able to be largely rescued by expressing NFS1Δ1-55/ISD11 but cells expressing NFS1Δ1-55/ISD11-R68L showed only a minor improvement in growth. The growth of E. coli was recorded by measuring the OD_600nm every 30 to 60 min. Data are represented as mean ± standard deviations determined from three independent experiments.

Figure 9.

A schematic model of Fe–S protein biosynthesis in the human mitochondria and cytosol. ISD11 forms a complex with NFS1, releasing sulfur (yellow circle) from cysteine. Iron (red circle) is imported from the cytosol facilitated by MFRN1/2 in the mitochondrial inner membrane. Although not fully understood, FXN is thought to be involved in transporting iron to the scaffold protein ISCU, while the NFS1-ISD11 complex delivers the sulfur. FXN also interacts with ISD11, and chaperone proteins HSPA9 (31) and HSCB (67). The clusters are then assembled into target apoproteins by a group of chaperone proteins HSCB, HSPA9 and GRPEL1/2, and GLRX5 with the recently proposed BOLA3 (9). Some Fe–S apoproteins require additional protein factors for ISC insertion, such as aconitase 2, which requires ISCA1, ISCA2 and IBA57, whereas OXPHOS complex I subunits require NUBPL. NFU1 is a specific scaffold protein that functions downstream of ISCU, and is required for ISC insertion in lipoic acid synthase and complex II subunit (25). ISCA1 and IBA57 are also required for this process (–70). Based on the studies done in yeast, it has been long believed that ABCB7 in the mitochondrial inner membrane plays a role in exporting an unknown product (X) from the mitochondrial ISC system (12,71). This export process is facilitated by GFER and GSH, and is essential for assembly of cytosolic Fe–S proteins. Proteins implicated in human disease are marked with an asterisk. The figure was modified from Sheftel et al. (3).