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Import and assembly of Neurospora crassa Tom40 into mitochondria of Trypanosoma brucei in vivo

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Abstract

The TOM complex (translocase of the mitochondrial outer membrane) is a dynamic, multisubunit protein complex. Tom40 is the major component of the complex and forms the preprotein conducting pore. To determine if a heterologous Tom40 could be properly targeted and assembled into the Trypanosoma brucei mitochondrial outer membrane, an ectopic copy of a gene encoding Neurospora crassa Tom40 (NcTom40) was expressed in procyclic trypanosomes from a tetracycline regulated procyclic acidic repetitive protein promoter. The level of NcTom40 expression was found to be maximal within 20–26 h of induction with tetracycline. Immunoblot analysis of subcellular fractions showed that NcTom40 was enriched in the mitochondrial fraction. Alkali extraction of isolated mitochondria revealed that NcTom40 was assembled as an integral membrane protein and limited proteolysis demonstrated that it was present in the outer membrane of the mitochondria. These data demonstrate that a heterologous mitochondrial protein containing internal targeting information can be correctly targeted to T. brucei mitochondria. Following blue native gel electrophoresis, the NcTom40 protein was found in a 370 kDa complex which may contain T. brucei Tom components. A 16 kDa protein was coimmunoprecipitated from T. brucei mitochondria containing NcTom40 using antisera developed against the N. crassa protein. The 16 kDa protein may represent a component of the T. brucei TOM complex that associates with NcTom40.

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Acknowledgements

We thank George Cross and Liz Wirtz from Rockefeller University for the pLew100 plasmid and the procyclic cell line 29–13, David Engman and Kevin Tyler for T. cruzi hsp70 antibody, and Stephen Hajduk for T. brucei ISP antibody. We also thank Lipi Saha for excellent technical assistance. Research was supported by NIH grants 5KO1 HL03839 and 3SO6GM08037–30S1.

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Authors and Affiliations

  1. Department of Microbiology, Meharry Medical College, Nashville, 37208 TN, USA

    Minu Chaudhuri

  2. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada

    Frank E. Nargang

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  1. Minu Chaudhuri
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  2. Frank E. Nargang
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Correspondence to Minu Chaudhuri.

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Communicated by M. Brunner

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Chaudhuri, M., Nargang, F.E. Import and assembly of Neurospora crassa Tom40 into mitochondria of Trypanosoma brucei in vivo. Curr Genet 44, 85–94 (2003). https://doi.org/10.1007/s00294-003-0427-y

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  • DOI: https://doi.org/10.1007/s00294-003-0427-y

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