Subcellular location of MMACHC and MMADHC, two human proteins central to intracellular vitamin B12 metabolism
Highlights
► MMACHC localizes to the cytoplasm by IF and subcellular fractionation. ► MMADHC localizes to the mitochondria and cytoplasm by IF and subcellular fractionation. ► Transduced GFP-tagged proteins rescue biochemical defects in MMA and/or HC.
Introduction
Vitamins are essential nutrients required for normal growth, development, and function in mammals. Acquired from the diet, vitamin B12 (cobalamin) is metabolized within the cell into two physiologically relevant forms, methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) [1]. Defects in intracellular vitamin B12 metabolism were originally identified by studying patients with rare, inherited defects in this pathway, resulting in the identification of nine complementation groups: cblA-cblG, cblJ, and mut [2], [3]. Inborn errors affecting synthesis of AdoCbl result in a decreased function of the mitochondrial enzyme methylmalonyl CoA mutase (MUT) and accumulation of its substrate, methylmalonic acid. Inborn errors affecting methylcobalamin result in a decreased activity of the cytoplasmic enzyme methionine synthase (MS) and accumulation of its substrate homocysteine. Defects in early steps of the pathway that are common to the synthesis of both cobalamin coenzymes result in accumulation of methylmalonic acid and homocysteine. Methylmalonic acidemia/aciduria (MMA) is associated with episodes of life-threatening metabolic acidemia and long-term development of neurologic and renal problems. Hyperhomocysteinemia and homocystinuria (HC) are associated with megaloblastic anemia and a variety of neurologic defects [4]. Inborn errors of cobalamin metabolism may present with isolated MMA, isolated HC, or combined MMA and HC depending upon which step in the pathway is affected.
The subcellular locations of MS and MUT play important roles in the metabolism and dispersion of vitamin B12 throughout the cell. MS functions in the cytoplasm, whereas MUT resides in the mitochondrion [5], [6]. The cblC and cblD complementation groups exhibit unique biochemical phenotypes. cblC presents exclusively as combined MMA and HC whereas cblD patients can have three distinct phenotypes: isolated homocystinuria (cblD-HC), isolated methylmalonic aciduria (cblD-MMA), or combined homocystinuria and methylmalonic aciduria (cblD-MMA/HC) [7], [8]. Based on phenotypic presentation and the known subcellular localization of MS and MUT, it is thought that MMACHC functions in the cytoplasm upstream of MMADHC. MMADHC would then act as a branch point for vitamin B12 delivery between the cytoplasm and mitochondria. Further, MMACHC and MMADHC have been shown to complex in vivo and in vitro [9], [10]. Genotype–phenotype correlation analysis of the three cblD variants suggests that the N- and C-termini of MMADHC have specific functions in the mitochondrion and cytoplasm, respectively [7], [11], [12]. Mutations in cblD-MMA patients result in premature translation termination at the protein level before Met62 or Met116. Reinitiation of translation at one of these residues could produce truncated versions of MMADHC lacking the N-terminal residues. These MMADHC isoforms would permit cytoplasmic MS function but lack mitochondrial MUT function. In contrast, cblD-HC patients have mutations at conserved residues at the C-terminus but retain integrity of N-terminal residues proposed to be involved in mitochondrial MUT function. Finally, cblD-MMA/HC patients have mutations after Met116, creating severely truncated proteins. In summary, cblD-MMA retains cytoplasmic function due to translation of an error-free C-terminus facilitated by downstream reinitiation; cblD-HC retains mitochondrial function with a full-length protein which harbors only C-terminal missense mutations in conserved residues; and in cblD-MMA/HC complete loss of functionality occurs with nonsense or splicing mutations that are localized downstream of Met116.
Here we report the subcellular location of MMACHC and MMADHC as determined from overexpressing GFP (green fluorescent protein)-fusions in transduced fibroblasts and from endogenous protein in control fibroblasts. To study the localization of proposed MMADHC isoforms, truncated MMADHC corresponding to translation initiation at Met62 (MMADHC∆1-61) was also studied. Subcellular localization was determined by immunofluorescence (IF) imaging and subcellular fractionation with immunoblotting, and the functionality of the fusion proteins was determined by biochemical complementation analysis.
Section snippets
Cloning
cDNA sequences of MMACHC and MMADHC were cloned into the pEGFP-N1 vector (Clontech) via restriction digestion and ligation using XhoI and BamHI restriction sites to incorporate a C-terminal GFP. MMACHC-GFP, MMADHC-GFP and MMADHC∆1-61-GFP were subsequently PCR amplified to incorporate attB1 and attB2 recombination sites for Gateway cloning (Invitrogen). Oligonucleotide primers are shown in Supplementary Table 1. Gene fusions were then cloned into the Gateway-modified retroviral mammalian
Subcellular location of MMACHC and of MMADHC isoforms by immunofluorescence
To investigate the subcellular distribution of MMACHC and MMADHC we first constructed retroviral vectors expressing GFP-fusion proteins and verified their expression by immunoblot analysis using an anti-GFP antibody (Fig. 1). The molecular masses of the GFP-fusions were consistent with values calculated from amino acid sequence: MMACHC-GFP: 59.4 kDa; MMADHC-GFP: 60.6 kDa; MMADHCΔ1-61-GFP: 54.2 kDa. Free GFP was not detected in the lysates of the cells expressing the fusion constructs. We
Discussion
Reports of the subcellular localization of the MMACHC protein have been equivocal. On the basis of the cblC cellular phenotype, it is recognized as a cytoplasmic protein. Functionally, MMACHC lies downstream of the LMBD1 and ABCD4 proteins that apparently mediate transfer of endocytosed cobalamin across the lysosomal membrane and into the cytoplasm [2]. MMACHC lies upstream of the MMADHC protein, proposed to partition internalized cobalamin between the cytoplasmic MS and mitochondrial MUT
Acknowledgments
This research was supported by operating grants to J.W.C. from the Natural Sciences and Engineering Research Council (NSERC; RGPIN 7289-06-07), to D.S.R. from the Canadian Institutes of Health Research (CIHR; MOP-15078), and to E.A.S. from the Canadian Institutes of Health Research (CIHR; Mt-15460). We thank Timothy Johns for cell-line immortalizations and imaging of MMACHC-HA and MMACHC-FLAG. Tamiko Nishimura provided tissue culture training. Jaeseung Kim contributed to the initial project
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