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Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy.

Liu HY, Liao PC, Chuang KT, Kao MC - J. Biomed. Sci. (2011)

Bottom Line: A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein.The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan, People's Republic of China.

ABSTRACT

Background: NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclear-encoded subunits existing in human mitochondrial complex I. Defects in this subunit have been associated with Parkinson's disease, Alzheimer's disease, Bipolar disorder, and Schizophrenia. The aim of this study is to examine the mitochondrial targeting of NDUFV2 and dissect the pathogenetic mechanism of one human deletion mutation present in patients with early-onset hypertrophic cardiomyopathy and encephalopathy.

Methods: A series of deletion and point-mutated constructs with the c-myc epitope tag were generated to identify the location and sequence features of mitochondrial targeting sequence for NDUFV2 in human cells using the confocal microscopy. In addition, various lengths of the NDUFV2 N-terminal and C-terminal fragments were fused with enhanced green fluorescent protein to investigate the minimal region required for correct mitochondrial import. Finally, a deletion construct that mimicked the IVS2+5_+8delGTAA mutation in NDUFV2 gene and would eventually produce a shortened NDUFV2 lacking 19-40 residues was generated to explore the connection between human gene mutation and disease.

Results: We identified that the cleavage site of NDUFV2 was located around amino acid 32 of the precursor protein, and the first 22 residues of NDUFV2 were enough to function as an efficient mitochondrial targeting sequence to carry the passenger protein into mitochondria. A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein. The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.

Conclusions: The mitochondrial targeting sequence of NDUFV2 is located at the N-terminus of the precursor protein. Maintaining a net positive charge and an amphiphilic structure with the overall balance and distribution of basic and hydrophobic amino acids in the N-terminus of NDUFV2 is important for mitochondrial targeting. The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

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Effects of hydrophobic residue mutation in NDUFV2 MTS on mitochondrial targeting. (a) The sites of hydrophobic residue in NDUFV2 N-terminal 1-32 amino acids were underlined and marked. (b) The effect of hydrophobic residue mutation within the N-terminal region of NDUFV2 on mitochondrial targeting. A series of point mutations targeting at hydrophobic residues were introduced into NDUFV2 with the c-myc epitope tag and expressed in T-REx-293 cells. The expressed proteins with hydrophobic residue mutations were labeled by an anti-c-myc-FITC antibody in transfected cells (green color), mitochondria were labeled by Mito Tracker Red (red color), and colocalization of expressed protein and mitochondria is shown as a merged image and indicated by yellow signals. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization. The (++++) symbol indicates all of the FITC fluorescence signals in transfected cells are fully colocalized with mitochondria. The (-) symbol indicates that there is no cell producing FITC fluorescence within the mitochondrial compartment. Scale bars = 10  μm.
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Figure 7: Effects of hydrophobic residue mutation in NDUFV2 MTS on mitochondrial targeting. (a) The sites of hydrophobic residue in NDUFV2 N-terminal 1-32 amino acids were underlined and marked. (b) The effect of hydrophobic residue mutation within the N-terminal region of NDUFV2 on mitochondrial targeting. A series of point mutations targeting at hydrophobic residues were introduced into NDUFV2 with the c-myc epitope tag and expressed in T-REx-293 cells. The expressed proteins with hydrophobic residue mutations were labeled by an anti-c-myc-FITC antibody in transfected cells (green color), mitochondria were labeled by Mito Tracker Red (red color), and colocalization of expressed protein and mitochondria is shown as a merged image and indicated by yellow signals. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization. The (++++) symbol indicates all of the FITC fluorescence signals in transfected cells are fully colocalized with mitochondria. The (-) symbol indicates that there is no cell producing FITC fluorescence within the mitochondrial compartment. Scale bars = 10 μm.

Mentions: To examine the effect of basic, hydrophobic and hydroxylated residues within the N-terminal region of NDUFV2 on mitochondrial targeting, a site-directed mutagenesis methodology was applied systematically on these three groups of residues. The positively charged arginine, lysine and histidine residues were changed to non-charged residues, hydrophobic residues were replaced with hydrophilic residues and hydroxylated residues were substituted with residues without a hydroxyl group. The N-terminal 1-32 amino acids of NDUFV2 contain eight basic residues, including Arg8, Arg10, His17, Arg20, His21, Arg23, His26 and Lys27 (Figure 6a). Surprisingly, none of the substitutions at each individual basic amino acid residue affected the mitochondrial targeting function of the protein (data not shown). When three arginine residues (Arg8, Arg10 and Arg20) and one histidine (His17) were mutated at the same time to generate a quadruple mutant (Figure 6b), the resulting protein still yielded a mitochondrial localization pattern indistinguishable from that of the wild-type NDUFV2. However, when the fifth amino acid substitution (H21A) was introduced into the quadruple mutant, a slight reduction in the mitochondrial targeting was observed in the resulting protein (Figure 6b). With the introduction of increasing number of mutations in the basic residues, the resulting mutant gradually lost its capability of mitochondrial import. When all of the eight basic residues were mutated at the same time (the R8G+R10A+H17A+R20A+H21A+R23A+H26A+K27A octuple mutant), the ability of mitochondrial targeting of the protein was almost completely destroyed (Figure 6b). To further confirm the result obtained from confocal images, the strategy of subcellular fractionation, followed with quantitative analyses by Western blots was also applied on several mutants with a single-pointed mutation or multiple-pointed mutations on the basic residues. As shown in Figure 6c, the quantitative signals for the single-pointed mutant (R23A), quintuple mutant (R8G+R10A+H17A+R20A+H21A) and sextuple mutant (R8G+R10A+H17A+R20A+H21A+R23A) were 92%, 74% and 22%, respectively, of those of the wild-type T-REx-293 cell. This result is corresponding very well with the data derived from aforementioned confocal image analyses. Interestingly, when the same mutagenesis approach was applied to investigate the role of hydrophobic residues in the MTS of NDUFV2, a similar phenomenon was observed. Eight hydrophobic residues in total, including Phe2, Phe3, Leu7, Leu14, Trp18, Val22, Leu25 and Ala29 (Figure 7a), were selected for mutation to evaluate the effects of these changes on mitochondrial import but all of the single-point mutants showed an import efficiency comparable to that of the wild-type NDUFV2 (data not shown). A clear deficiency in mitochondrial targeting of these mutants was started to be observed when five hydrophobic residues in NDUFV2 N-terminus were mutated (the L7Q+L14Q+V22G+ L25Q +A29G quintuple mutant shown in Figure 7b). When 7 hydrophobic residues were mutated simultaneously (the L7Q+L14Q+V22G+ L25Q +A29G+W18Y+F3Y septuple mutant) the mitochondrial localization pattern was completely abolished. Finally, the only three hydroxylated residues, including Ser4, Thr15 and Thr28 in the NDUFV2 presequence were used for mutation, and the result showed that all of the mutations including single-, double- and triple-point mutations did not have a significant effect on the mitochondrial targeting of this protein (data not shown).


Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy.

Liu HY, Liao PC, Chuang KT, Kao MC - J. Biomed. Sci. (2011)

Effects of hydrophobic residue mutation in NDUFV2 MTS on mitochondrial targeting. (a) The sites of hydrophobic residue in NDUFV2 N-terminal 1-32 amino acids were underlined and marked. (b) The effect of hydrophobic residue mutation within the N-terminal region of NDUFV2 on mitochondrial targeting. A series of point mutations targeting at hydrophobic residues were introduced into NDUFV2 with the c-myc epitope tag and expressed in T-REx-293 cells. The expressed proteins with hydrophobic residue mutations were labeled by an anti-c-myc-FITC antibody in transfected cells (green color), mitochondria were labeled by Mito Tracker Red (red color), and colocalization of expressed protein and mitochondria is shown as a merged image and indicated by yellow signals. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization. The (++++) symbol indicates all of the FITC fluorescence signals in transfected cells are fully colocalized with mitochondria. The (-) symbol indicates that there is no cell producing FITC fluorescence within the mitochondrial compartment. Scale bars = 10  μm.
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Related In: Results  -  Collection

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Figure 7: Effects of hydrophobic residue mutation in NDUFV2 MTS on mitochondrial targeting. (a) The sites of hydrophobic residue in NDUFV2 N-terminal 1-32 amino acids were underlined and marked. (b) The effect of hydrophobic residue mutation within the N-terminal region of NDUFV2 on mitochondrial targeting. A series of point mutations targeting at hydrophobic residues were introduced into NDUFV2 with the c-myc epitope tag and expressed in T-REx-293 cells. The expressed proteins with hydrophobic residue mutations were labeled by an anti-c-myc-FITC antibody in transfected cells (green color), mitochondria were labeled by Mito Tracker Red (red color), and colocalization of expressed protein and mitochondria is shown as a merged image and indicated by yellow signals. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization. The (++++) symbol indicates all of the FITC fluorescence signals in transfected cells are fully colocalized with mitochondria. The (-) symbol indicates that there is no cell producing FITC fluorescence within the mitochondrial compartment. Scale bars = 10 μm.
Mentions: To examine the effect of basic, hydrophobic and hydroxylated residues within the N-terminal region of NDUFV2 on mitochondrial targeting, a site-directed mutagenesis methodology was applied systematically on these three groups of residues. The positively charged arginine, lysine and histidine residues were changed to non-charged residues, hydrophobic residues were replaced with hydrophilic residues and hydroxylated residues were substituted with residues without a hydroxyl group. The N-terminal 1-32 amino acids of NDUFV2 contain eight basic residues, including Arg8, Arg10, His17, Arg20, His21, Arg23, His26 and Lys27 (Figure 6a). Surprisingly, none of the substitutions at each individual basic amino acid residue affected the mitochondrial targeting function of the protein (data not shown). When three arginine residues (Arg8, Arg10 and Arg20) and one histidine (His17) were mutated at the same time to generate a quadruple mutant (Figure 6b), the resulting protein still yielded a mitochondrial localization pattern indistinguishable from that of the wild-type NDUFV2. However, when the fifth amino acid substitution (H21A) was introduced into the quadruple mutant, a slight reduction in the mitochondrial targeting was observed in the resulting protein (Figure 6b). With the introduction of increasing number of mutations in the basic residues, the resulting mutant gradually lost its capability of mitochondrial import. When all of the eight basic residues were mutated at the same time (the R8G+R10A+H17A+R20A+H21A+R23A+H26A+K27A octuple mutant), the ability of mitochondrial targeting of the protein was almost completely destroyed (Figure 6b). To further confirm the result obtained from confocal images, the strategy of subcellular fractionation, followed with quantitative analyses by Western blots was also applied on several mutants with a single-pointed mutation or multiple-pointed mutations on the basic residues. As shown in Figure 6c, the quantitative signals for the single-pointed mutant (R23A), quintuple mutant (R8G+R10A+H17A+R20A+H21A) and sextuple mutant (R8G+R10A+H17A+R20A+H21A+R23A) were 92%, 74% and 22%, respectively, of those of the wild-type T-REx-293 cell. This result is corresponding very well with the data derived from aforementioned confocal image analyses. Interestingly, when the same mutagenesis approach was applied to investigate the role of hydrophobic residues in the MTS of NDUFV2, a similar phenomenon was observed. Eight hydrophobic residues in total, including Phe2, Phe3, Leu7, Leu14, Trp18, Val22, Leu25 and Ala29 (Figure 7a), were selected for mutation to evaluate the effects of these changes on mitochondrial import but all of the single-point mutants showed an import efficiency comparable to that of the wild-type NDUFV2 (data not shown). A clear deficiency in mitochondrial targeting of these mutants was started to be observed when five hydrophobic residues in NDUFV2 N-terminus were mutated (the L7Q+L14Q+V22G+ L25Q +A29G quintuple mutant shown in Figure 7b). When 7 hydrophobic residues were mutated simultaneously (the L7Q+L14Q+V22G+ L25Q +A29G+W18Y+F3Y septuple mutant) the mitochondrial localization pattern was completely abolished. Finally, the only three hydroxylated residues, including Ser4, Thr15 and Thr28 in the NDUFV2 presequence were used for mutation, and the result showed that all of the mutations including single-, double- and triple-point mutations did not have a significant effect on the mitochondrial targeting of this protein (data not shown).

Bottom Line: A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein.The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan, People's Republic of China.

ABSTRACT

Background: NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclear-encoded subunits existing in human mitochondrial complex I. Defects in this subunit have been associated with Parkinson's disease, Alzheimer's disease, Bipolar disorder, and Schizophrenia. The aim of this study is to examine the mitochondrial targeting of NDUFV2 and dissect the pathogenetic mechanism of one human deletion mutation present in patients with early-onset hypertrophic cardiomyopathy and encephalopathy.

Methods: A series of deletion and point-mutated constructs with the c-myc epitope tag were generated to identify the location and sequence features of mitochondrial targeting sequence for NDUFV2 in human cells using the confocal microscopy. In addition, various lengths of the NDUFV2 N-terminal and C-terminal fragments were fused with enhanced green fluorescent protein to investigate the minimal region required for correct mitochondrial import. Finally, a deletion construct that mimicked the IVS2+5_+8delGTAA mutation in NDUFV2 gene and would eventually produce a shortened NDUFV2 lacking 19-40 residues was generated to explore the connection between human gene mutation and disease.

Results: We identified that the cleavage site of NDUFV2 was located around amino acid 32 of the precursor protein, and the first 22 residues of NDUFV2 were enough to function as an efficient mitochondrial targeting sequence to carry the passenger protein into mitochondria. A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein. The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.

Conclusions: The mitochondrial targeting sequence of NDUFV2 is located at the N-terminus of the precursor protein. Maintaining a net positive charge and an amphiphilic structure with the overall balance and distribution of basic and hydrophobic amino acids in the N-terminus of NDUFV2 is important for mitochondrial targeting. The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

Show MeSH
Related in: MedlinePlus