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Mitochondrial dysfunction confers resistance to multiple drugs in Caenorhabditis elegans.

Zubovych IO, Straud S, Roth MG - Mol. Biol. Cell (2010)

Bottom Line: Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2.Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant.Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCepsilon.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9038, USA.

ABSTRACT
In a previous genetic screen for Caenorhabditis elegans mutants that survive in the presence of an antimitotic drug, hemiasterlin, we identified eight strong mutants. Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2. Here we identify two additional mutations that confer drug resistance, spg-7 and har-1, also in genes encoding mitochondrial proteins. Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant. Respiratory complex inhibitors, FCCP and oligomycin, and a producer of reactive oxygen species (ROS), paraquat, all rescued wild-type worms from hemiasterlin toxicity. Worms lacking mitochondrial superoxide dismutase (MnSOD) were modestly drug-resistant, and elimination of MnSOD in the phb-2, har-1, and spg-7 mutants enhanced resistance. The antioxidant N-acetyl-l-cysteine prevented mitochondrial inhibitors from rescuing wild-type worms from hemiasterlin and sensitized mutants to the toxin, suggesting that a mechanism sensitive to ROS is necessary to trigger drug resistance in C. elegans. Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCepsilon.

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PHB-2 and CHCHD2 (Har-1) mutants localize to mitochondria. Hela cells were transfected with plasmid expression vectors expressing myc-tagged human PHB-2, human PHB-2(E130K), CHCHD2 (the human ortholog of Har-1), and CHCHD2(G65E). After 18 h cells were stained with MitoTracker Red, fixed, and stained with anti-myc antibody (green). PHB2 is known to localize to and CHCHD2 suspected of localizing to mitochondria. Each of the mutants also colocalized with the MitoTracker Red. In some cells such as the one shown for PHB-2 wild type, the myc-staining pattern resembled MitoTracker staining, but no MitoTracker was visible in the cells.
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Figure 2: PHB-2 and CHCHD2 (Har-1) mutants localize to mitochondria. Hela cells were transfected with plasmid expression vectors expressing myc-tagged human PHB-2, human PHB-2(E130K), CHCHD2 (the human ortholog of Har-1), and CHCHD2(G65E). After 18 h cells were stained with MitoTracker Red, fixed, and stained with anti-myc antibody (green). PHB2 is known to localize to and CHCHD2 suspected of localizing to mitochondria. Each of the mutants also colocalized with the MitoTracker Red. In some cells such as the one shown for PHB-2 wild type, the myc-staining pattern resembled MitoTracker staining, but no MitoTracker was visible in the cells.

Mentions: PHB-2 and SPG-7 proteins are known to localize to mitochondria in several species, but the subcellular location of HAR-1 had not been reported. CHCHD2 is the human ortholog of HAR-1. We obtained cDNA for CHCHD2 and introduced into it the mutation found in C. elegans har-1. Wild-type and mutant myc-tagged CHCHD2, with wild-type and mutant PHB-2 for comparison, were expressed transiently in HeLa cells, and the staining pattern with c-myc antibody was compared with MitoTracker Red staining (Figure 2). Both wild-type and mutant CHCHD2 localized exclusively to mitochondria as indicated by the colocalization with MitoTracker Red. For both CHCHD2 and PHB-2, some cells stained only with the c-myc antibody and had no MitoTracker staining (wild-type PHB-2, Figure 2). In these cells the reticular c-myc staining became more punctate, a result that would occur if mitochondria lost membrane potential and fragmented in those cells. We also observed that transient transfection appeared cytotoxic after longer periods, suggesting that overexpressing these proteins was toxic.


Mitochondrial dysfunction confers resistance to multiple drugs in Caenorhabditis elegans.

Zubovych IO, Straud S, Roth MG - Mol. Biol. Cell (2010)

PHB-2 and CHCHD2 (Har-1) mutants localize to mitochondria. Hela cells were transfected with plasmid expression vectors expressing myc-tagged human PHB-2, human PHB-2(E130K), CHCHD2 (the human ortholog of Har-1), and CHCHD2(G65E). After 18 h cells were stained with MitoTracker Red, fixed, and stained with anti-myc antibody (green). PHB2 is known to localize to and CHCHD2 suspected of localizing to mitochondria. Each of the mutants also colocalized with the MitoTracker Red. In some cells such as the one shown for PHB-2 wild type, the myc-staining pattern resembled MitoTracker staining, but no MitoTracker was visible in the cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2836976&req=5

Figure 2: PHB-2 and CHCHD2 (Har-1) mutants localize to mitochondria. Hela cells were transfected with plasmid expression vectors expressing myc-tagged human PHB-2, human PHB-2(E130K), CHCHD2 (the human ortholog of Har-1), and CHCHD2(G65E). After 18 h cells were stained with MitoTracker Red, fixed, and stained with anti-myc antibody (green). PHB2 is known to localize to and CHCHD2 suspected of localizing to mitochondria. Each of the mutants also colocalized with the MitoTracker Red. In some cells such as the one shown for PHB-2 wild type, the myc-staining pattern resembled MitoTracker staining, but no MitoTracker was visible in the cells.
Mentions: PHB-2 and SPG-7 proteins are known to localize to mitochondria in several species, but the subcellular location of HAR-1 had not been reported. CHCHD2 is the human ortholog of HAR-1. We obtained cDNA for CHCHD2 and introduced into it the mutation found in C. elegans har-1. Wild-type and mutant myc-tagged CHCHD2, with wild-type and mutant PHB-2 for comparison, were expressed transiently in HeLa cells, and the staining pattern with c-myc antibody was compared with MitoTracker Red staining (Figure 2). Both wild-type and mutant CHCHD2 localized exclusively to mitochondria as indicated by the colocalization with MitoTracker Red. For both CHCHD2 and PHB-2, some cells stained only with the c-myc antibody and had no MitoTracker staining (wild-type PHB-2, Figure 2). In these cells the reticular c-myc staining became more punctate, a result that would occur if mitochondria lost membrane potential and fragmented in those cells. We also observed that transient transfection appeared cytotoxic after longer periods, suggesting that overexpressing these proteins was toxic.

Bottom Line: Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2.Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant.Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCepsilon.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9038, USA.

ABSTRACT
In a previous genetic screen for Caenorhabditis elegans mutants that survive in the presence of an antimitotic drug, hemiasterlin, we identified eight strong mutants. Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2. Here we identify two additional mutations that confer drug resistance, spg-7 and har-1, also in genes encoding mitochondrial proteins. Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant. Respiratory complex inhibitors, FCCP and oligomycin, and a producer of reactive oxygen species (ROS), paraquat, all rescued wild-type worms from hemiasterlin toxicity. Worms lacking mitochondrial superoxide dismutase (MnSOD) were modestly drug-resistant, and elimination of MnSOD in the phb-2, har-1, and spg-7 mutants enhanced resistance. The antioxidant N-acetyl-l-cysteine prevented mitochondrial inhibitors from rescuing wild-type worms from hemiasterlin and sensitized mutants to the toxin, suggesting that a mechanism sensitive to ROS is necessary to trigger drug resistance in C. elegans. Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCepsilon.

Show MeSH
Related in: MedlinePlus