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First published online 21 May 2008
doi: 10.1242/jcs.024034

Journal of Cell Science 121, 1990-1998 (2008)
Published by The Company of Biologists 2008
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Integration of tail-anchored proteins into the mitochondrial outer membrane does not require any known import components

Christian Kemper1, Shukry J. Habib1, Gertraud Engl2, Petra Heckmeyer1, Kai S. Dimmer2 and Doron Rapaport2,*

1 Institut für Physiologische Chemie der Universität München, Butenandtstr. 5, 81377 Munich, Germany
2 Interfakultäres Institut für Biochemie, Hoppe-Seyler-Str. 4, University of Tübingen, 72076 Tübingen, Germany


Figure 1
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  		Fig. 1. The TMD of Fis1 is sufficient for mitochondrial targeting. (A) Schematic representation of the GFP-Fis1(TM) protein. (B) Mitochondria (M) were purified via sucrose gradient from wild-type yeast cells or from cells transformed with a vector encoding GFP-Fis1(TM). The post mitochondrial fraction was further separated by differential centrifugation into ER fraction and cytosol (C). Proteins were subjected to SDS-PAGE and immunocytochemistry with antibodies against the GFP moiety, a control marker protein for the cytosol (Bmh1), a control ER marker protein (Erv2) and the mitochondrial outer-membrane protein Tom20.

 

Figure 2
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  		Fig. 2. Protection of Fis1-TMC from modification with IASD represents proper insertion into the outer membrane. (A) The sequences of the tail domains of native Fis1 and its variants. Only relevant residues in the cytosolic domain are indicated. The putative transmembrane segment is underlined. Cysteine and positively charged residues in the wild-type sequence and their replacement in the variant proteins are in bold type. (B) Fis1-TMC can complement the mitochondrial morphology phenotype of the fis1{Delta} strain. Cells of the indicated strains (containing mitochondria-targeted RFP) were analyzed by fluorescence (left) and phase-contrast (right) microscopy. (C) Endogenous Fis1-TMC is protected from modification with IASD. The indicated amounts of mitochondria isolated from a fis1{Delta} strain transformed with Fis1-TMC were incubated where indicated with IASD in the presence or absence of Triton X-100 (Tx-100). Mitochondrial proteins were analyzed by SDS-PAGE and immunocytochemistry with antibodies against Fis1. (D) Radiolabeled Fis1-TMC is protected from modification with IASD. Wild-type mitochondria were incubated with either radiolabeled Fis1 or radiolabeled Fis1-TMC for 30 minutes at 25°C. Mitochondria were re-isolated by centrifugation and resuspended in either import buffer or labeling buffer containing, where indicated, IASD and Tx-100. Mitochondrial proteins were analyzed by SDS-PAGE and autoradiography. (E) An insertion-deficient Fis1 variant is not protected from modification with IASD. Wild-type mitochondria were incubated with either radiolabeled Fis1-TMC or Fis1(TMC-4Q) for 30 minutes at 25°C. Further treatment was as described in D. (F) The modification of Fis1-TMC is cysteine specific. Radiolabeled precursors of Fis1(CS) and Fis1-TMC were analyzed directly by SDS-PAGE or were incubated for 20 minutes at 25°C in labeling buffer containing, where indicated, IASD and Tx-100. Further treatment was as described in D. A band representing hemoglobin, which is present at high amounts in the reticulocyte lysate is indicated with an asterisk.

 

Figure 3
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  		Fig. 3. The insertion of Fis1 does not require TOM import receptors. (A) Radiolabeled precursor of Fis1-TMC was incubated with intact mitochondria or mitochondria pretreated with trypsin. Modification with IASD was as described above. Mitochondrial proteins were separated by SDS-PAGE, blotted to a membrane and then analyzed by autoradiography and immunocytochemistry. The antibodies used were directed against the receptor proteins Tom70 and Tom20 and the membrane-embedded Tom40, where a small fragment is cleaved off upon trypsin treatment. Lower panel: as a control, radiolabeled porin was incubated with the intact or trypsin-pretreated mitochondria for the indicated time periods. Mitochondria were treated with proteinase K (100 µg/ml) to degrade non-inserted protein and mitochondrial proteins were analyzed by SDS-PAGE and autoradiography. Insertion of Fis1 was calculated as the fraction of bound material (–IASD), which is protected in the presence of IASD (lower band in +IASD). The amount of precursor imported into intact mitochondria after 20 minutes was set to 100%. (B) Radiolabeled Fis1-TMC was incubated with mitochondria isolated from either the wild type or from cells lacking Tom20. Lower panel: as a control, radiolabeled porin was incubated with the same mitochondria and further treatment and analysis was as described in A. (C) Radiolabeled Fis1-TMC was incubated with mitochondria isolated from either the wild type or from cells lacking Tom70. Lower panel: as a control, radiolabeled porin was incubated with the same mitochondria and further treatment and analysis was as described in A. (D) Radiolabeled Fis1-TMC was incubated at 0°C for the indicated time points with either wild-type mitochondria or with mitochondria isolated from cells lacking either Tom20 or Tom70. Further treatment was as described in the legend to Fig. 2D. The bands were quantified and the intensity of the band corresponding to the unmodified protein was taken as a measure for protein insertion. The amount of protein inserted into mitochondria isolated from the corresponding wild-type strain after initial incubation for 20 minutes was set at 100%.

 

Figure 4
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  		Fig. 4. Known import components in the outer membrane are not required for the insertion of Fis1. (A) Radiolabeled precursor of Fis1-TMC was incubated with isolated mitochondria in the presence or absence of excess recombinant matrix-destined precursor pSu9-DHFR. Further modification with IASD was as described in Fig. 2D. Lower panel: as a control, radiolabeled porin was incubated with isolated mitochondria in the presence or absence of excess pSu9-DHFR for the indicated time periods. Mitochondria were treated with proteinase K and mitochondrial proteins were analyzed by SDS-PAGE and autoradiography. (B) Radiolabeled Fis1-TMC was incubated with either wild-type mitochondria or with mitochondria isolated from cells harboring a temperature-sensitive allele of Tom40 (tom40-3) or lacking Tom6 (tom6{Delta}). Further treatment was as described in the legend to Fig. 2D. Lower panel, as a control, radiolabeled pSu9-DHFR was incubated with the indicated type of mitochondria for various time periods. The band corresponding to the mature form was quantified and the amount of precursor protein imported into control mitochondria after incubation for 20 minutes was set to 100%. Note that each mutant strain has different parental wild type strain. The precursor and mature forms of pSu9-DHFR are indicated with p and m, respectively. (C) Radiolabeled Fis1-TMC was incubated with either wild-type mitochondria or with mitochondria isolated from cells lacking Mas37 (mas37{Delta}). Further treatment was as described in Fig. 2D. (D) Experiments similar to that presented in Fig. 4B,C were performed with the indicated mutated strains and their corresponding wild-type strain. For each strain at least three independent experiments were performed. The bands corresponding to the inserted protein (unmodified protein in the presence of IASD, lower band in +IASD) were quantified. For each mutated strain the amount of protein inserted into the mutated mitochondria was compared with that inserted into mitochondria isolated from the corresponding wild-type strain, which was set to 100%. The error bars represent s.d.

 

Figure 5
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  		Fig. 5. High ergosterol content reduces the efficiency of insertion of Fis 1 into the membrane of lipid vesicles. (A) Radiolabeled precursor of Fis1-TMC was incubated for the indicated time periods with either mitochondria (50 µg protein) or an equivalent amount of lipid vesicles (33 µg lipids). Further treatment was as described in Fig. 2D. (B) Radiolabeled precursor of Fis1-TMC was incubated with lipid vesicles containing the indicated mol% of ergosterol. Further treatment was as described in Fig. 2D. The band that represents unlabeled Fis1 molecules in the presence of IASD (lower band in +IASD) was quantified by densitometry. Insertion was calculated as the intensity of this band in comparison to the amount of precursor protein added to the reaction. (C) Radiolabeled precursor of Tom20ext (Ahting et al., 2005Go) was incubated for 20 minutes at 25°C with either mitochondria or an equivalent amount of lipid vesicles containing the indicated mol% of ergosterol. After incubation, the samples were halved and in one half mitochondria or liposomes were pelleted and solubilized directly in sample buffer (–proteinase K). The other aliquots were treated with PK (500 µg/ml) before solubilization in sample buffer (+proteinase K). The specific membrane-inserted proteolytic fragment (Ahting et al., 2005Go) is indicated with an arrowhead. (D) Radiolabeled precursor of Fis1-TMC was incubated with a mixture of mitochondria (50 µg protein) and lipid vesicles (33 µg). Modification with IASD was as described in Fig. 2D. Mitochondria were separated from lipid vesicles by differential centrifugation and proteins were analyzed by SDS-PAGE and autoradiography. The bands were quantified and the intensity of the band corresponding to the unmodified protein was taken as a measure for protein insertion. For each mixture, the amount of protein inserted into mitochondria was set to 100%.

 

Figure 6
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  		Fig. 6. A charge variant of Fis1 is inserted into microsomes but not into the membrane of mitochondria or lipid vesicles. (A) Radiolabeled precursor of Fis1(TMC-4Q) was incubated for 20 minutes at 25°C with either mitochondria (50 µg protein) or lipid vesicles (33 µg lipids). Further treatment was as described in the legend to Fig. 2D. (B) Radiolabeled precursors of either Fis1-TMC or Fis1(TMC-4Q) were incubated for 20 minutes at 25°C with 50 µg of either mitochondria or microsomes. Further treatment was as described in Fig. 2D.

 

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© The Company of Biologists Ltd 2008