spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Suzuki, H.
Right arrow Articles by Mihara, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Suzuki, H.
Right arrow Articles by Mihara, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Characterization of rat TOM70 as a receptor of the preprotein translocase of the mitochondrial outer membrane

Hiroyuki Suzuki, Maki Maeda and Katsuyoshi Mihara*

Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan



View larger version (41K):

[in a new window]
  		Fig. 2. Subcellular localization of rTOM70 in rat liver cells. (A) Comparison of SDS-PAGE profiles of mitochondrial authentic OM70 (Mt) and the in vitro synthesized OM70 (TL) as detected by autoradiography and immunoblotting, respectively. (B) 50 µg each of mitochondria (mit), microsomes (ms) and cytosol (cyt) proteins from the rat liver were subjected to SDS-PAGE and immunoblotting using antibodies against the indicated proteins. The immunoreacted bands were visualized by ECL (Amersham). (C) Immunofluorescence detection of TOM70 in HeLa cells. HeLa cells were incubated with MitoTracker at 37°C for 20 minutes, fixed, stained with rabbit IgGs against rTOM70 and then stained with FITC-conjugated goat antibodies against rabbit. Images were obtained and analyzed using confocal fluorescence microscopy. (D) Submitochondrial localization of rTOM70. Submitochondrial fractionation by sucrose density gradient centrifugation was performed as described in the Materials and Methods. After centrifugation, the samples were fractionated from the top of the tubes. The fractions were TCA-precipitated and subjected to SDS-PAGE and then the proteins were detected by immunoblotting with antibodies against the indicated proteins. Inp represents 20 µg of the unfractionated sample.

 


View larger version (57K):

[in a new window]
  		Fig. 1. Comparison of the deduced amino-acid sequences of Tom70 from rats and N. crassa. (A) The sequences of rats and N. crassa Tom70 were aligned using the Multiple Sequence program (DNASIS). The amino-acid residues conserved among species were shaded, TMDs were boxed and TPR motifs were underlined and numbered. TPR motifs were searched for using the Protein Families database alignment program (Pfam). (B) Comparison of hydropathy profiles of Tom70 from rats and N. crassa using the Kyte and Doolittle algorithm at a span setting of 10 amino-acid residues.

 


View larger version (43K):

[in a new window]
  		Fig. 3. Characterization of rTOM70 as a mitochondrial integral membrane protein. (A) Mitochondria were treated with either 100 mM Na2CO3 (pH 11.5) or 1 M KCl at 0°C for 30 minutes. The reaction mixtures were centrifuged to separate the supernatant (S) and precipitate (P) fractions, and each fraction was subjected to SDS-PAGE followed by immunoblot analysis using antibodies against the indicated proteins. (B) Rat liver mitochondria were treated with the indicated concentrations of trypsin at 0°C for 20 minutes and then soybean trypsin inhibitor was added to the reaction mixtures and incubated at 0°C for 20 minutes. The reaction mixtures were centrifuged to separate the supernatant and precipitate fractions, and each fraction was resolved by SDS-PAGE and analyzed by immunoblotting with the antibodies against the indicated proteins.

 


View larger version (40K):

[in a new window]
  		Fig. 4. Effect of antibodies against rTOM70 on mitochondrial preprotein import.

(A) Mitochondria were preincubated with the indicated concentrations of preimmune IgG (PI), anti-rTOM70 IgG ({alpha}70) or anti-rTOM20 IgG ({alpha}20) at 0°C for 30 minutes, reisolated and subjected to the import of pre-Su9-DHFR, pAd, AAC and rTOM40. When import of AAC or rTOM40 was assayed, the reaction mixtures were treated with proteinase K at 0°C for 30 minutes under hypotonic (for AAC) or isotonic (for rTOM40) conditions after import. After the reaction, 1 mM PMSF was added to the reaction mixtures, the mixture was centrifuged, the precipitates were resolved by SDS-PAGE and the gels were analyzed by FLA2000. Other conditions were as described in the Materials and Methods. The import efficiencies were expressed relative to the control import in the absence of IgG or in the presence of preimmune IgG. Where indicated, the import reactions were performed in the presence of 10 µM CCCP. (B) Effect of anti-rTOM70 IgGs on the MSF- or reticulocyte-lysate-supported mitochondrial import of pAd. 35S-pAd or 125I-pAd was subjected to the import reaction in the presence of MSFS (right) or rabbit reticulocyte lysate (left), respectively, using the mitochondria that had been treated with the indicated IgGs (200 µg/ml each) and reisolated. After the reaction, the reaction mixtures were resolved by SDS-PAGE and the gels were analyzed by autoradiography. Other conditions were as described in Materials and Methods.

 


View larger version (30K):

[in a new window]
  		Fig. 5. rTOM70 is loosely associated with the ~400 kDa complex containing rTOM22 and rTOM40. (A) Interaction of rTOM70 with the import components of the outer membrane as probed by coimmunoprecipitation. The outer membranes were solubilized with 2% digitonin-50 mM NaCl, and aliquots of the supernatant were subjected to immunoprecipitation at 4°C for 3 hours using preimmune, anti-rTOM20, anti-rTOM40 or anti-rTOM70 IgGs. The immunoprecipitates were resolved by SDS-PAGE and the proteins were visualized by immunoblotting with antibodies against the indicated proteins. Inp represents 20% of an aliquot of the supernatant fraction. Other conditions were as described in the Materials and Methods. (B) Analysis of rat TOM proteins with blue native PAGE. Rat liver mitochondrial outer membranes were solubilized with 2% digitonin-containing buffer, and solubilized supernatant was subjected to blue native PAGE as described in the Materials and Methods. Gel slots were excised and subjected to the second dimensional Tricine SDS-PAGE. The gels were analyzed by immunoblotting with the indicated IgGs. Marker proteins used were serum albumin, 66 kDa; lactate dehydrogenase, 140 kDa; catalase, 232 kDa; apoferritin, 440 kDa; and thyroglobulin, 669 kDa. (C) Interaction of rTOM70 with rTOM22 and rTOM20 as revealed by the yeast two-hybrid assay. Host strain Y190 was transformed with two plasmids, one encoding the GAL4 DNA-binding domain (BD) fused to the cytosolic domain of either rTOM20 (BD-rTOM20) or rTOM22 (BD-rTOM22) and the other encoding the GAL4-activating domain (AD) fused to the cytosolic domains of the import components (AD-rTOM20, AD-rTOM22, AD-rTOM70, or AD-OM37). Positive interactions were verified by growth of the transformants on synthetic complete medium without histidine (upper panel) or ß-galactosidase activity (lower panel). As a control, the host strain was transformed with an empty vector encoding AD (pACT2).

 


View larger version (45K):

[in a new window]
  		Fig. 6. Characterization of the mitochondria-targeting signal of rTOM70. (A) Basic amino acids at the C-terminal flanking region of the TMD are critical for targeting rTOM70. COS-7 cells were transfected with the indicated constructs in the expression vectors. After 24 hours, the cells were incubated with Mito Tracker, then the cells were fixed and immunostained with IgG against rabbit polyclonal anti-HA epitope tag antibodies and FITC-conjugated antibodies against rabbit IgG. To monitor the localization of rTOM70S6HA, cells were treated with 5 µg/ml BFA for 1 hour, fixed and immunostained with rabbit polyclonal IgGs against rat Calnexin and FITC-conjugated antibodies against rabbit IgGs. To detect localization of the GFP fusions in COS-7 cells, the cells were co-stained with Mito Tracker, and fluorescence images were obtained using a confocal microscope. (B) Binding and insertion of rTOM70HA, rTOM70S6HA or rTOM70(1-69)GFP into mitochondria in vitro. Reticulocyte-lysate-synthesized 35S-labeled proteins were subjected to mitochondrial import. To measure mitochondria binding of these proteins, the reaction mixtures were centrifuged to isolate mitochondria (P) and the supernatant (S). To verify membrane integration (`Import'), the reaction mixtures were treated with 100 mM sodium carbonate (pH 11.5) at 0°C for 30 minutes and centrifuged to separate the supernatant (S) and membrane (P) fractions. Both fractions were subjected to SDS-PAGE and subsequent fluoroimage analysis. (C) Membrane integration of rTOM70 into trypsin-treated mitochondria. Mitochondrial import assay of rTOM70 was performed using 20 µg/ml trypsin-treated or untreated mitochondria as described in (B). An aliquot of sodium-carbonate-treated mitochondria was centrifuged to separate the membrane (P) and supernatant (S) fractions, and both fractions were subjected to immunoblot analysis using the antibodies against the indicated proteins.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002