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

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.