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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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The N-terminal 22-amino acid region of NDUFV2 is essential and efficient for mitochondrial targeting. (a) The diagrammatic representation of EGFP fusion proteins carrying an NDUFV2 N-terminal peptide of a different length. A series of chimeric cDNA were constructed for expression of fusion proteins containing the full-length (NDUFV21-249 -EGFP), N-terminal (NDUFV21-32 -EGFP, NDUFV21-22 -EGFP, NDUFV21-21 -EGFP, NDUFV21-20 -EGFP, NDUFV21-18 -EGFP) or internal fragment (NDUFV28-22 -EGFP) in the MTS of NDUFV2 with EGFP at the C-terminus or at the N-terminus (EGFP-NDUFV21-22). The number of (+) symbols indicates the relative number of cells that exhibited EGFP fluorescence within the mitochondrial compartment in (b). The number of (+) symbols indicates that the proportion of cells exhibiting EGFP fluorescence have a typical punctuated staining pattern and mitochondrial colocalization in (b). The (++++) symbol indicates all of the EGFP fluorescence signals in transfected cells are fully colocalized with mitochondria. The (-) symbol indicates there is no cell producing EGFP fluorescence within the mitochondrial compartment. (b) The distribution of EGFP fusion proteins in transfected T-REx-293 cells was detected by EGFP fluorescence 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-I are corresponding to constructs A-I shown in (a). Scale bars = 10  μm.
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Figure 4: The N-terminal 22-amino acid region of NDUFV2 is essential and efficient for mitochondrial targeting. (a) The diagrammatic representation of EGFP fusion proteins carrying an NDUFV2 N-terminal peptide of a different length. A series of chimeric cDNA were constructed for expression of fusion proteins containing the full-length (NDUFV21-249 -EGFP), N-terminal (NDUFV21-32 -EGFP, NDUFV21-22 -EGFP, NDUFV21-21 -EGFP, NDUFV21-20 -EGFP, NDUFV21-18 -EGFP) or internal fragment (NDUFV28-22 -EGFP) in the MTS of NDUFV2 with EGFP at the C-terminus or at the N-terminus (EGFP-NDUFV21-22). The number of (+) symbols indicates the relative number of cells that exhibited EGFP fluorescence within the mitochondrial compartment in (b). The number of (+) symbols indicates that the proportion of cells exhibiting EGFP fluorescence have a typical punctuated staining pattern and mitochondrial colocalization in (b). The (++++) symbol indicates all of the EGFP fluorescence signals in transfected cells are fully colocalized with mitochondria. The (-) symbol indicates there is no cell producing EGFP fluorescence within the mitochondrial compartment. (b) The distribution of EGFP fusion proteins in transfected T-REx-293 cells was detected by EGFP fluorescence 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-I are corresponding to constructs A-I shown in (a). Scale bars = 10 μm.

Mentions: After identification of the MTS in NDUFV2 as well as the probable cleavage site of the protein, this study attempted to define the minimal region required for mitochondrial targeting. A series of chimeric constructs for expression of NDUFV2 MTS-EGFP fusion protein were generated (Figure 4a). As shown in Figure 4b, EGFP along without any targeting sequence addition was present throughout the cell, with some accumulation in the nucleus. On the other hand, EGFP fused with the full-length NDUFV21-249 or the newly identified MTS (NDUFV21-32) colocalized very well with that of the Mito Tracker Red dye, indicating that the first 32 amino acid acids in the N-terminus of NDUFV2 had a mitochondrial targeting ability comparable to that of the full-length NDUFV2. It was interesting to observe that the protein fragment containing the first 22 amino acid residues of NDUFV2 was sufficient to carry most (if not all) of the EGFP into mitochondria successfully, whereas the regions containing either the first 21 (NDUFV21-21 -EGFP) or 20 (NDUFV21-20 -EGFP) amino acid residues in the N-terminal sequence of NDUFV2 showed a much lower efficiency. When the first 18 amino acid residues were used as the signal peptide, the majority of mitochondrial targeting ability of this hybrid protein was lost (Figure 4b). The NDUFV28-22-EGFP was also incapable of targeting to mitochondria. Together, these results indicate that the entire 1-22 residues are necessary for mitochondria targeting of NDUFV2.


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)

The N-terminal 22-amino acid region of NDUFV2 is essential and efficient for mitochondrial targeting. (a) The diagrammatic representation of EGFP fusion proteins carrying an NDUFV2 N-terminal peptide of a different length. A series of chimeric cDNA were constructed for expression of fusion proteins containing the full-length (NDUFV21-249 -EGFP), N-terminal (NDUFV21-32 -EGFP, NDUFV21-22 -EGFP, NDUFV21-21 -EGFP, NDUFV21-20 -EGFP, NDUFV21-18 -EGFP) or internal fragment (NDUFV28-22 -EGFP) in the MTS of NDUFV2 with EGFP at the C-terminus or at the N-terminus (EGFP-NDUFV21-22). The number of (+) symbols indicates the relative number of cells that exhibited EGFP fluorescence within the mitochondrial compartment in (b). The number of (+) symbols indicates that the proportion of cells exhibiting EGFP fluorescence have a typical punctuated staining pattern and mitochondrial colocalization in (b). The (++++) symbol indicates all of the EGFP fluorescence signals in transfected cells are fully colocalized with mitochondria. The (-) symbol indicates there is no cell producing EGFP fluorescence within the mitochondrial compartment. (b) The distribution of EGFP fusion proteins in transfected T-REx-293 cells was detected by EGFP fluorescence 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-I are corresponding to constructs A-I 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 4: The N-terminal 22-amino acid region of NDUFV2 is essential and efficient for mitochondrial targeting. (a) The diagrammatic representation of EGFP fusion proteins carrying an NDUFV2 N-terminal peptide of a different length. A series of chimeric cDNA were constructed for expression of fusion proteins containing the full-length (NDUFV21-249 -EGFP), N-terminal (NDUFV21-32 -EGFP, NDUFV21-22 -EGFP, NDUFV21-21 -EGFP, NDUFV21-20 -EGFP, NDUFV21-18 -EGFP) or internal fragment (NDUFV28-22 -EGFP) in the MTS of NDUFV2 with EGFP at the C-terminus or at the N-terminus (EGFP-NDUFV21-22). The number of (+) symbols indicates the relative number of cells that exhibited EGFP fluorescence within the mitochondrial compartment in (b). The number of (+) symbols indicates that the proportion of cells exhibiting EGFP fluorescence have a typical punctuated staining pattern and mitochondrial colocalization in (b). The (++++) symbol indicates all of the EGFP fluorescence signals in transfected cells are fully colocalized with mitochondria. The (-) symbol indicates there is no cell producing EGFP fluorescence within the mitochondrial compartment. (b) The distribution of EGFP fusion proteins in transfected T-REx-293 cells was detected by EGFP fluorescence 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-I are corresponding to constructs A-I shown in (a). Scale bars = 10 μm.
Mentions: After identification of the MTS in NDUFV2 as well as the probable cleavage site of the protein, this study attempted to define the minimal region required for mitochondrial targeting. A series of chimeric constructs for expression of NDUFV2 MTS-EGFP fusion protein were generated (Figure 4a). As shown in Figure 4b, EGFP along without any targeting sequence addition was present throughout the cell, with some accumulation in the nucleus. On the other hand, EGFP fused with the full-length NDUFV21-249 or the newly identified MTS (NDUFV21-32) colocalized very well with that of the Mito Tracker Red dye, indicating that the first 32 amino acid acids in the N-terminus of NDUFV2 had a mitochondrial targeting ability comparable to that of the full-length NDUFV2. It was interesting to observe that the protein fragment containing the first 22 amino acid residues of NDUFV2 was sufficient to carry most (if not all) of the EGFP into mitochondria successfully, whereas the regions containing either the first 21 (NDUFV21-21 -EGFP) or 20 (NDUFV21-20 -EGFP) amino acid residues in the N-terminal sequence of NDUFV2 showed a much lower efficiency. When the first 18 amino acid residues were used as the signal peptide, the majority of mitochondrial targeting ability of this hybrid protein was lost (Figure 4b). The NDUFV28-22-EGFP was also incapable of targeting to mitochondria. Together, these results indicate that the entire 1-22 residues are necessary for mitochondria targeting of NDUFV2.

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