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First published online 27 July 2004
doi: 10.1242/jcs.01275

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Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones

Takunari Yoneda^*,, Cristina Benedetti^*, Fumihiko Urano, Scott G. Clark, Heather P. Harding and David Ron^¶

Skirball Institute of Biomolecular Medicine and the Departments of Cell Biology and Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA

View larger version (54K): [in a new window]   Fig. 1. Attenuated function of the mitochondrial genome induces mtHSP70 (hsp-6) mRNA. (A) Autoradiogram of a Southern blot of EcoRI-digested total DNA from animals raised in the indicated concentration of ethidium bromide (EtBr; µg/ml), or tunicamycin (Tun; 1 µg/ml). The blot was hybridized sequentially to a labeled DNA fragment of the mitochondrially encoded cox-1 gene and the nuclear encoded K08H10.2a gene. (B) Autoradiogram of the northern blot of total RNA from animals cultured on agar plates containing the indicated concentration of ethidium bromide or tunicamycin. (Upper panel) The blot was hybridized to a labeled cox-1 DNA fragment. (Lower panel) Ethidium bromide staining of the ribosomal bands of the RNA. (C) Autoradiogram of a northern blot of total RNA from animals treated with ethidium bromide, tunicamycin or heat-shock (HS). The blot was hybridized sequentially to radiolabled fragment from the mtHSP70 gene (hsp-6) and the C. elegans BiP homologue, hsp-4. Bar charts on the right of each panel show the relative intensity of the hybridization signal.

View larger version (45K): [in a new window]   Fig. 2. Reporter genes activated by mitochondrial perturbation. (A) Fluorescence photomicrographs of untreated (UT), ethidium bromide (EtBr; µg/ml) or tunicamycin-treated (Tun; 1 µg/ml) or heat-shocked (HS) animals with reporter transgenes for mitochondrial chaperones (hsp-6::gfp, hsp-60::gfp), an endoplasmic reticulum chaperone (hsp-4::gfp), a cytoplasmic chaperone (hsp-70::gfp), and the mitochondrial tri-carboxylic acid cycle enzymes citrate synthetase (cts-1::gfp) and aconitase (aco-2::gfp). (B) Immunoblot of soluble proteins extracted from the hsp-6::gfp animals described in A. The blot was reacted with anti-GFP serum (upper panel) or antiserum to the broadly expressed UNC-32 protein (lower panel). (C) Northern blot of untreated and ethidium bromide-treated hsp-6::gfp animals. The blot was hybridized sequentially with a hsp-6-coding region probe that detects the endogenous gene and a GFP probe that detects the transgene.

View larger version (61K): [in a new window]   Fig. 5. Activation of mitochondrial chaperone genes can be uncoupled from depolarization of the mitochondrial membrane potential and the accumulation of reactive oxygen species. (A) Fluorescence photomicrographs of animals cultured on plates containing the indicated concentration of dinitrophenol (DNP, mM) and subsequently exposed to the fluorescent dye tetramethylrhodamine ethyl ester (TMRE). (B) Immunoblot of GFP (upper panel) or UNC-32 (lower panel) in lysates from untreated (UT), dinitrophenol (DNP; mM), ethidium bromide (EtBr, 125 µg/ml), spg-7(RNAi), tunicamycin (Tun; 1 µg/ml) treated or heat shocked (HS) animals with hsp-6::gfp, hsp-60::gfp and hsp-4::gfp reporters. (C) Autoradiogram of a northern blot or Southern blot of total RNA or DNA from untreated animals (UT), dinitrophenol (DNP; mM) or ethidium bromide (EtBr, 125 µg/ml) treatment. The blots were hybridized to a radiolabeled fragment from the C. elegans mitochondrially encoded cox-1 gene or nuclear hsp-6 or KO8H10.2a genes. (D) Fluorescent photomicrographs of hsp-60::gfp and sod-3::gfp transgenic animals following RNAi of genes involved in mitochondrial protein processing or exposure to the toxin Paraquat (2 mM).

View larger version (58K): [in a new window]   Fig. 3. Interference with genes that process mitochondrial proteins selectively activates mitochondrial chaperone genes. The top row of panels shows photomicrographs of wild-type (N2) animals exposed to the indicated RNAi; the panels below are photomicrographs of GFP fluorescence of hsp-6::gfp, hsp-60::gfp, hsp-4::gfp, hsp-70::gfp, cts-1::gfp and aco-2::gfp animals exposed to mock RNAi or RNAi of the indicated genes.

View larger version (40K): [in a new window]   Fig. 4. Interference with spg-7 (predicted to encode a mitochondrial protease) or ethidium bromide treatment perturbs protein processing in the mitochondria and activate the endogenous hsp-6 gene. (A) Immunoblot of GFP in detergent extracts from transgenic animals expressing conventional GFP (a cytoplasmic protein) or GFP with an N-terminal mitochondrial import sequence (mtGFP), both in the body wall muscle. The animals were exposed to mock RNAi or spg-1(RNAi) or cultured on 125 µg/ml ethidium bromide (EtBr) as indicated. Equal fractions of the total extract (Total), 100,000 g soluble (Soluble) and 100,000 g pellet fractions (Pellet) of the whole animal detergent extract were loaded on the gel as indicated. (B) Autoradiogram of a northern blot of total RNA from mock-RNAi, spg-7(RNAi) or ethidium bromide (EtBr) treated animals. The blot was hybridized sequentially to radiolabeled fragments from the hsp-6, spg-7, aco-2, cts-1, F1F0 ATP synthase -chain (F1F0) and cytochrome c oxidase subunit IV (Cyt C Ox IV) genes. (C) Quantification of the radiolabeled signal on the blots shown in B. The hybridization signal for each gene at the untreated point was set as 1.