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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 NDUFV2 N-terminal and C-terminal truncation on mitochondrial targeting of the protein. (a) The constructs generated to express full-length and truncated NDUFV2 proteins. Full-length NDUFV2 (A), N-terminal truncation (B, △1-18 NDUFV2; C, △1-32 NDUFV2; D, △1-50 NDUFV2) and C-terminal truncation (E, △198-249 NDUFV2; F, △183-249 NDUFV2) were fused with c-myc epitope tag, and expressed in T-REx-293 cells. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization in (b). 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. (b) The distribution of c-myc fusion proteins was detected by anti-c-myc-FITC antibody (green color) and mitochondria were labeled by Mito Tracker Red (red color). Only merged images are shown (colocalization of expressed protein and mitochondria is indicated by yellow signals). Photos A-F are corresponding to constructs A-F shown in (a). Scale bars = 10  μm.
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Figure 2: Effects of NDUFV2 N-terminal and C-terminal truncation on mitochondrial targeting of the protein. (a) The constructs generated to express full-length and truncated NDUFV2 proteins. Full-length NDUFV2 (A), N-terminal truncation (B, △1-18 NDUFV2; C, △1-32 NDUFV2; D, △1-50 NDUFV2) and C-terminal truncation (E, △198-249 NDUFV2; F, △183-249 NDUFV2) were fused with c-myc epitope tag, and expressed in T-REx-293 cells. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization in (b). 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. (b) The distribution of c-myc fusion proteins was detected by anti-c-myc-FITC antibody (green color) and mitochondria were labeled by Mito Tracker Red (red color). Only merged images are shown (colocalization of expressed protein and mitochondria is indicated by yellow signals). Photos A-F are corresponding to constructs A-F shown in (a). Scale bars = 10 μm.

Mentions: NDUFV2 is a nuclear-encoded mitochondrial protein which is assembled into the L-shaped complex I and is localized in the hydrophilic arm protruding into the matrix. Therefore, this protein is expected to be imported into mitochondria through a pathway specific for mitochondrial matrix proteins. Analyses of this protein by MitoProt II [35] suggested a 99.6% probability of mitochondrial targeting of NDUFV2. A very similar result was also obtained from the prediction from the TargetP server [36]. Protein sequence alignment of NDUFV2 from various species revealed that the proteins from eukaryotic species have a non-conserved region located at the N-terminus (Figure 1a). It has been predicted that the first 32 amino acids of NDUFV2 may be the MTS of this protein [32]. To test this prediction, full-length, various N-terminal and C-terminal deletion constructs were generated to determine the location and orientation of MTS in NDUFV2 (Figure 2a). A c-myc epitope tag was appended to the C-terminus of these constructs to facilitate detection and analysis by the immunofluorescent staining method. All of the designed constructs were successfully engineered from the NDUFV2 cDNA and confirmed by direct sequencing. After transient transfection, mouse anti-c-myc antibody was applied to detect the expressed proteins, and the Mito Tracker Red dye was used to mark the mitochondria in T-REx-293 cells. The results showed that the full-length NDUFV2 construct had a punctuated cytosolic staining pattern that was typically observed when mitochondria were immunostained, indicating applicable of the experimental strategy. When the C-terminal deletion constructs (pcDNA4-△183-249 NDUFV2 and pcDNA4-△198-249 NDUFV2) were individually transfected into T-REx-293 cells, both of the truncated proteins were still colocalized with mitochondria. However, N-terminal truncations of NDUFV2 including △1-18 NDUFV2, △1-32 NDUFV2 and △1-50 NDUFV2, all lost their mitochondrial localization (Figure 2b). These observations agree well with the suggestion from protein sequence alignment and the protein domain prediction programs, and indicate that the MTS of NDUFV2 is located at the N-terminus of the precursor protein.


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 NDUFV2 N-terminal and C-terminal truncation on mitochondrial targeting of the protein. (a) The constructs generated to express full-length and truncated NDUFV2 proteins. Full-length NDUFV2 (A), N-terminal truncation (B, △1-18 NDUFV2; C, △1-32 NDUFV2; D, △1-50 NDUFV2) and C-terminal truncation (E, △198-249 NDUFV2; F, △183-249 NDUFV2) were fused with c-myc epitope tag, and expressed in T-REx-293 cells. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization in (b). 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. (b) The distribution of c-myc fusion proteins was detected by anti-c-myc-FITC antibody (green color) and mitochondria were labeled by Mito Tracker Red (red color). Only merged images are shown (colocalization of expressed protein and mitochondria is indicated by yellow signals). Photos A-F are corresponding to constructs A-F shown in (a). Scale bars = 10  μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117770&req=5

Figure 2: Effects of NDUFV2 N-terminal and C-terminal truncation on mitochondrial targeting of the protein. (a) The constructs generated to express full-length and truncated NDUFV2 proteins. Full-length NDUFV2 (A), N-terminal truncation (B, △1-18 NDUFV2; C, △1-32 NDUFV2; D, △1-50 NDUFV2) and C-terminal truncation (E, △198-249 NDUFV2; F, △183-249 NDUFV2) were fused with c-myc epitope tag, and expressed in T-REx-293 cells. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization in (b). 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. (b) The distribution of c-myc fusion proteins was detected by anti-c-myc-FITC antibody (green color) and mitochondria were labeled by Mito Tracker Red (red color). Only merged images are shown (colocalization of expressed protein and mitochondria is indicated by yellow signals). Photos A-F are corresponding to constructs A-F shown in (a). Scale bars = 10 μm.
Mentions: NDUFV2 is a nuclear-encoded mitochondrial protein which is assembled into the L-shaped complex I and is localized in the hydrophilic arm protruding into the matrix. Therefore, this protein is expected to be imported into mitochondria through a pathway specific for mitochondrial matrix proteins. Analyses of this protein by MitoProt II [35] suggested a 99.6% probability of mitochondrial targeting of NDUFV2. A very similar result was also obtained from the prediction from the TargetP server [36]. Protein sequence alignment of NDUFV2 from various species revealed that the proteins from eukaryotic species have a non-conserved region located at the N-terminus (Figure 1a). It has been predicted that the first 32 amino acids of NDUFV2 may be the MTS of this protein [32]. To test this prediction, full-length, various N-terminal and C-terminal deletion constructs were generated to determine the location and orientation of MTS in NDUFV2 (Figure 2a). A c-myc epitope tag was appended to the C-terminus of these constructs to facilitate detection and analysis by the immunofluorescent staining method. All of the designed constructs were successfully engineered from the NDUFV2 cDNA and confirmed by direct sequencing. After transient transfection, mouse anti-c-myc antibody was applied to detect the expressed proteins, and the Mito Tracker Red dye was used to mark the mitochondria in T-REx-293 cells. The results showed that the full-length NDUFV2 construct had a punctuated cytosolic staining pattern that was typically observed when mitochondria were immunostained, indicating applicable of the experimental strategy. When the C-terminal deletion constructs (pcDNA4-△183-249 NDUFV2 and pcDNA4-△198-249 NDUFV2) were individually transfected into T-REx-293 cells, both of the truncated proteins were still colocalized with mitochondria. However, N-terminal truncations of NDUFV2 including △1-18 NDUFV2, △1-32 NDUFV2 and △1-50 NDUFV2, all lost their mitochondrial localization (Figure 2b). These observations agree well with the suggestion from protein sequence alignment and the protein domain prediction programs, and indicate that the MTS of NDUFV2 is located at the N-terminus of the precursor protein.

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