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First published online 15 July 2008
doi: 10.1242/jcs.026625

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Characterization of the mitochondrial protein LETM1, which maintains the mitochondrial tubular shapes and interacts with the AAA-ATPase BCS1L

¹ Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan
² Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan
³ Section of Functional Morphology, Faculty of Pharmaceuitical Science, Nagasaki International University, Nagasaki, Japan
⁴ Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo, Japan
⁵ The Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan

View larger version (45K): [in this window] [in a new window]   Fig. 1. LETM1 is a mitochondrial inner-membrane protein. (A) Total cell lysates from HeLa cells transfected with vector (lane 1), LETM1 (lane 2) or LETM1-3HA expression plasmid (lane 3) were subjected to immunoblotting using anti-LETM1 antibody. Gray and black arrowheads indicate precursor and mature forms of LETM1, respectively. (B) HeLa cells were fixed, permeabilized and immunostained with antibody against LETM1 (a) and the mitochondrial matrix protein Hsp60 (b). Images were acquired using confocal microscopy. Scale bar, 10 µm. (C) Total lysate (T) from HeLa cells carrying the LETM2 expression plasmid was fractionated and separated into mitochondria-enriched (Mt), microsomal (Ms) and cytosolic (Cyto) fractions. These fractions were subjected to immunoblotting using antibodies against LETM1, LETM2, the mitochondrial outer-membrane protein Tom40, the mitochondrial inner-membrane protein Tim17, the ER protein Sec61β and the cytosolic protein H450. Gray and solid arrowheads indicate precursor and mature forms of LETM1, respectively. (D) The mitochondrial fraction prepared from HeLa cells was treated at 4°C for 30 minutes with trypsin at the indicated concentrations with (lanes 4, 8) or without 1% Triton X-100 (lanes 1-3, 5-7) under isotonic (Mitochondria) or hypotonic (Mitoplasts) conditions and analyzed by immunoblotting with antibodies against LETM1, Tim17, Hsp60 and the outer membrane protein Tom20.

View larger version (50K): [in this window] [in a new window]   Fig. 2. LETM1 downregulation causes mitochondrial swelling. (A) Total-cell lysates from HeLa cells transfected once or twice with siRNA for either GFP (mock) or LETM1 were subjected to immunoblotting with antibodies to LETM1 and GAPDH as a loading control. (B) HeLa cells that stably expressed mitochondrion-targeted Su9-DsRed were transfected with siRNA targeting either GFP (a, control) or LETM1 (b). Then, the expression plasmid, which coexpressed LETM1 and nucleus-targeted GFP, was introduced into the cells transfected with the LETM1-targeting siRNA (c). Live images were obtained by confocal microscopy. Typical images were superimposed. Scale bar, 10 µm. (C) Cells transfected with siRNA (as described for B) were analyzed by immunofluorescence microscopy. Data represent the mean ± s.e. of three independent experiments; 100-200 individual cells were counted. (D) HeLa cells expressing Su9-DsRed were transfected with siRNAs targeting DRP1 (a), DRP1 and LETM1 (b), OPA1 (e), OPA1 and LETM1 (f), or LETM1 (panel d). After siRNA transfection, the plasmid expressing Drp1(K38A)-EGFP was transfected (c,d). Live images were collected by confocal microscopy. Arrowheads indicate large dot structures on the filaments, asterisks indicate cells expressing Drp1-EGFP. Scale bar, 10 µm. Data represent the mean ± s.e. of three independent experiments; 100-200 individual cells were counted. (E) HeLa cells transfected with siRNA targeting GFP (a-d) or LETM1 (e-h) were fixed, permeabilized and immunostained using antibodies against the inner membrane protein mitofilin and the matrix protein mtHsp70. Scale bar, 10 µm. (F) HeLa cells transfected with siRNA targeting either GFP (a, control) or LETM1 (b,c) were fixed and thin sections were visualized by electron microscopy. Arrowheads indicate swollen mitochondria. Scale bar, 950 nm. (G) Cells stably expressing Su9-DsRed were transfected with plasmid expressing LETM1-EGFP and analyzed by confocal microscopy. Inset, sigh-magnification image. Arrowhead indicates the cell overexpressing LETM1-EGFP. Scale bar, 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 3. LETM2 is a mitochondrial protein that is expressed preferentially in testis and sperm. (A) Scheme of LETM1, LETM2, and LETM2S. Red, blue, orange and green boxes represent putative presequences, hydrophobic segments, leucine-zipper motifs and EF-hand motifs, respectively. (B) Poly(A) RNA from adult rat tissues was hybridized using a radioactive probe recognizing LETM2. The blot was also hybridized with a control probe recognizing β-actin. (C) Total-cell lysates from various rat tissues were subjected to immunoblotting using antibodies against LETM2, LETM1 and GAPDH. (D) Total cell lysates from the indicated cell lines were analyzed by immunoblotting using antibodies against LETM2, LETM1 and Hsp60. (E) Total-cell lysates from HeLa cells transfected with vector (lane 1), or with plasmid expressing LETM2 (lane 2) or LETM2S (lane 3), and rat testis homogenates (lane 4) were subjected to immunoblotting using anti-LETM2 antibody. Black and gray arrowheads indicate LETM2 and LETM2S, respectively. (F) HeLa cells transfected with LETM2 (a-c) or LETM2S expression plasmid (d-f) were fixed, permeabilized and immunostained with antibodies against LETM2 and cytochrome c. Images were collected using confocal microscopy. Scale bar, 10 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 4. LETM2 is localized in spermatogenic cells and spermatozoa. (A) Frozen sections of rat testis were immunostained using anti-LETM2 antibody (a,b,e,f) and preimmune serum (c,d). Samples were also counterstained using SYTOX Green to visualize nuclear DNA (b,d,f). Scale bar, 30 µm. (B) Purified rat spermatozoa were immunostained using antibodies against LETM2 (a,b) and tubulin (b,d). Images containing the principle (P) and middle pieces (M) of spermatozoa are shown. Scale bar, 20 µm. (C) Vertical sections (a) and cross sections (b,c) of rat spermatozoa were immunogold-labeled using anti-LETM2 antibody and preimmune serum. Arrows indicate mitochondria. A, axoneme; ODF, outer dense fiber. Scale bar, 0.1 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 5. LETM1 is required for the assembly of the respiratory chain. (A) HeLa cells transfected with siRNA targeting GFP (control) or LETM1 were incubated for 60 minutes with Mitotracker Red CMXRos at the indicated concentrations, washed, fixed and analyzed by immunofluorescence microscopy with anti-mitofilin antibody. The uptake of Mitotracker Red CMXRos clearly decreased in the LETM1 knockdown cells. Fluorescent images were obtained by confocal microscopy. (B) Total cell lysates from cells transfected with siRNA targeting GFP (control) or LETM1 were subjected to SDS-PAGE and immunoblotted using antibodies to the indicated proteins. LETM1 knockdown caused reduction in the supercomplexes of the respiratory chains. (C) Mitochondrial fractions from cells transfected with siRNA targeting GFP (control) or LETM1 were digitonin-solubilized, centrifuged, subjected to Blue-Native gel electrophoresis and immunoblotted with antibodies against the indicated proteins. Arrowhead indicates the supercomplexes of complexes I, III and IV. Asterisk indicates a nonspecific band that was found even when BCS1L was repressed (see Fig. 8B). (D) Mitochondrial fractions from cells transfected with siRNA targeting GFP (control) or LETM1 were treated with alkaline buffers ranging in their pH values between 11.5 and 12.5, centrifuged for separation into supernatant (S) and pellet fractions (P), and immunoblotted with antibodies against the indicated proteins. The membrane association of core1 protein was affected by LETM1 downregulation.

View larger version (42K): [in this window] [in a new window]   Fig. 6. Mitochondrial tubular networks are maintained in 0 HeLa cells. (A) Total cell lysates from wild-type or 0 HeLa cells were analyzed by immunoblotting using antibodies targeting the indicated proteins. 0 HeLa cells completely lost the supercomplexes. (B) Mitochondrial fractions from wild-type or 0 HeLa cells were digitonin-solubilized, subjected to Blue-Native gel electrophoresis, and immunoblotted with antibodies against the indicated proteins. Arrowhead indicates the supercomplexes of complexes I, III and IV. (C) The 0 HeLa cells transfected with the plasmid carrying Su9-DsRed were analyzed by confocal microscopy. 0 HeLa cells maintain mitochondrial tubules, which are not expanded. Scale bar, 10 µm. (D) The 0 HeLa cells were fixed, and thin sections were visualized by electron microscopy. Arrowheads indicate mitochondria. Scale bar, 950 nm.

View larger version (47K): [in this window] [in a new window]   Fig. 7. BCS1L interacts with LETM1 and stimulates the formation of the LETM1 complex. (A) Cell lysates from HeLa cells transfected with vector, BCS1L-3FLAG or AIF-3FALG expression plasmid were subjected to immunoprecipitation (Immppt) using anti-FLAG antibody. Precipitates were analyzed by immunoblotting using antibodies against the indicated proteins. HC, immunoglobulin heavy chains. LETM1 interacts with BCS1L. (B) Cell lysates from cells expressing wild-type BCS1L or the indicated BCS1L mutants were analyzed by immunoprecipitation (Immppt) using anti-FLAG antibody and immunoblotting. The mutation in BCS1L disrupted interaction with LETM1. (C) HeLa cells transfected with the expression plasmid carrying wild-type BCS1L or the indicated BCS1L mutants were fixed, permeabilized and immunostained using antibodies against FLAG (a,d,g,j,m,p,s) and the mitochondrial protein mtHsp70 (b,e,h,k,n,q,t). Untransfected HeLa cells were also analyzed by immunofluorescence microscopy using antibodies against BCS1L (v) and mtHsp70 (w). Scale bar, 10 µm. BCS1L mutations did not influence its mitochondrial localization. (D) HeLa cells transfected with BCS1L siRNA (lanes 1, 5, 9), GFP siRNA (lanes 2, 6, 10), or BCS1L expression plasmid (lanes 4, 8, 12) were analyzed by either SDS-PAGE (left panel) or Blue-Native gel electrophoresis (right panels) and immunoblotting using antibodies against LETM1, BCS1L and Tom40 as a loading control. Formation of the LETM1 complex was dependent on BCS1L.

View larger version (50K): [in this window] [in a new window]   Fig. 8. BCS1L downregulation causes supercomplex disassembly and abnormal mitochondrial morphology. (A) Total cell lysates from cells transfected with siRNA targeting GFP (control) or BCS1L were immunoblotted with antibodies to the indicated proteins. BCS1L knockdown caused reduction in the supercomplexes of the respiratory chains. (B) Mitochondrial fractions from cells transfected with siRNA targeting GFP (control) or BCS1L were solubilized with digitonin, subjected to Blue-Native gel electrophoresis, and immunoblotted using antibodies against the indicated proteins. Arrowhead indicates supercomplexes of Complex I, III, and IV. Asterisk indicates a nonspecific band. (C) HeLa cells that stably expressed Su9-DsRed were transfected with siRNA for GFP (panel a), BCS1L (panels b,c,d), LETM1 (panel e), or BCS1L and LETM1 (panel f), and further transfected with plasmid co-expressing BCS1L and nucleus-targeted GFP (panel b). BCS1L knockdown induced abnormal mitochondrial morphology. Live images were acquired by confocal microscopy. Typical images are shown in insets at high magnification. Scale bar, 10 µm. (D) Cells transfected with BCS1L siRNA were analyzed by immunofluorescence microscopy. Data represent the mean ± s.e. of three independent experiments; 100-200 individual cells were counted. (E) Model of the functions of BCS1L and LETM1 in mitochondrial biogenesis. Blue arrows indicate functions reported here; gray arrow indicates the BCS1L function reported previously (Cruciat et al., 1999; de Lonlay et al., 2001).

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