Here we describe the first human patients with mutations in MTFMT. The causal mutations were verified by rescuing the mitochondrial translation defects in patient fibroblasts via lentiviral transduction of MTFMT. Analysis of the tRNAMet pools in patient fibroblasts revealed severe MTFMT dysfunction. To our knowledge, this is the first time the human mitochondrial tRNAMet profile has been analyzed. It is interesting to note that control fibroblasts lack detectable Met-tRNAMet, suggesting that it is utilized as quickly as it is produced; either converted to fMet-tRNAMet or used to donate Met to the growing polypeptide chain. Strikingly, patient fibroblasts lack detectable levels of fMet-tRNAMet and contain mostly Met-tRNAMet.
Drastically decreased fMet-tRNAMet
levels prevent efficient mitochondrial translation as demonstrated by the reduced translation observed in patient fibroblasts. Although fibroblasts from P1 and P2 have severely impaired mitochondrial translation, they do retain some residual activity. To understand the origin of this activity, we measured the relative distribution of three possible N-terminal states of mitochondrially translated COX1 by mass spectrometry. While this is not the first targeted study of the N-termini of COX1 (Escobar-Alvarez et al., 2010
), to our knowledge, this is the first time all three states are measured, and the first observation of mitochondrial methionine excision activity, which is detectable albeit weak.
Formylated COX1 is the dominant species in patient fibroblasts, indicating residual MTFMT activity. Assuming P1's nonsense mutation has a full loss of function, then the allele harboring the shared c.626C>T mutation must confer MTFMT activity. Transcript that has not undergone skipping of exon 4 encodes an MTFMT variant harboring a p.S209L missense mutation. Residue p.S209 is moderately conserved and lies on the periphery of MTFMT based on homology with the bacterial enzyme.
Similarly, P2's residual MTFMT activity must originate from enzyme variants carrying the p.S209L mutation and/or the p.S125L mutation located in the active site.
Studies in bacteria and yeast have raised questions about the absolute requirement for Met-tRNAMet
formylation. Formylation is not essential in all bacteria (Newton et al., 1999
) and in yeast disruption of FMT1
causes no discernible defect in mitochondrial protein synthesis or function (Hughes et al., 2000
; Li et al., 2000
; Vial et al., 2003
). Additionally, bovine IF2mt
is able to restore respiration in a yeast mutant lacking both IF2mt
and FMT1 (Tibbetts, 2003), suggesting that bovine IF2mt
, like yeast IF2mt
, can initiate protein synthesis without fMet-tRNAMet
. However, a number of studies in mammals indicate that formylation of mitochondrial Met-tRNAMet
is required for translation initiation. Bovine IF2mt
has a 25-50 fold greater affinity for fMet-tRNAMet
than for Met-tRNAMet in vitro
(Spencer and Spremulli, 2004
) and 12 of the 13 bovine mtDNA-encoded proteins retain fMet at the N-terminus (Walker et al., 2009
). The presence of some Met instead of fMet at the N-terminus of COX1 from patient fibroblasts (
) could, however, suggest that unformylated Met-tRNAMet
can initiate mitochondrial translation although less efficiently than fMet-tRNAMet
One of the factors that could allow Met-tRNAMet
to initiate mitochondrial translation is its increased concentration in patient fibroblasts. In Salmonella typhimurium
, amplification of initiator tRNA genes compensates for a lack of methionyl-tRNA formyltransferase activity and allows translation initiation without formylation of the initiator tRNA (Nilsson et al., 2006
). The “up-regulation” of the mitochondrial tRNAMet
in patient fibroblasts (
) could, in principle, be a compensatory response due to limited fMet-tRNAMet
In summary, we have used MitoExome sequencing to identify MTFMT
as a gene underpinning combined OXPHOS deficiency associated with Leigh syndrome. We have shown that patient fibroblasts have a striking deficiency of fMet-tRNAMet
leading to impaired mitochondrial translation. Despite studies in yeast suggesting that MTFMT
is not essential for mitochondrial translation (Hughes et al., 2000
; Li et al., 2000
; Vial et al., 2003
), we show here that in humans this gene is required for efficient mitochondrial translation and function. More generally, this study demonstrates how MitoExome sequencing can reveal insights into basic biochemistry and the molecular basis of mitochondrial disease.