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Forschungsarbeit

Molecular mechanisms of protein import into human mitochondria

By Christina Schusdziarra (15.01.2014)

Mitochondria are essential organelles of the eukaryotic cell. They are required for many important processes, such as oxidative phosphorylation, heme synthesis, biogenesis of iron sulfer cluster and induction of apoptotic pathways. Roughly 190 mitochondrial proteins have been reported to be involved in human diseases [1]. It is crucial to investigate the biogenesis of mitochondria in humans to better understand the development of these human diseases, the mediation of apoptosis and thereby also the mechanisms underlying cancer development and treatment.

Since only 13 mitochondrial proteins are encoded by mitochondrial DNA, the biogenesis of mitochondria requires the import of more than thousand different proteins encoded in the nucleus and synthesized as precursor proteins in the cytosol. Import into mitochondria is mediated by highly complex translocation machineries in the mitochondrial membranes [2]. Almost all of our knowledge about these translocases was obtained from studies in baker’s yeast, S. cerevisiae. The TIM23 translocase, the translocation complex of the inner mitochondrial membrane, transports precursor proteins into and across the inner mitochondrial membrane. Two multiple membrane-spanning subunits, Tim23 and Tim17, form the translocation pore in the membrane and are connected to the receptor, Tim50,in the intermembrane space, and to the ATP-dependent import motor on the matrix side of the inner membrane. The import motor represents a specialized Hsp70 chaperone system which is tightly regulated by the action of the co-chaperones Tim14 and Tim16 [3].

In humans, homologues of most of the yeast translocase components have been identified. However, little is known about the exact mechanisms of mitochondrial protein import in higher eukaryotes. The presence of two homologues for components such as Tim17 and Tim14 already suggests a more complex composition and / or regulation of this process in humans compared to yeast [4].

We have recently identified MCJ (methylation controlled J-protein) as a stimulating co-chaperone in the import motor of the human TIM23 translocase [5]. MCJ has first been described to be differentially expressed in ovarian cancer cell lines and short term cultures of normal ovarian epithelial cells [6]. The expression was dependent on the methylation status of the CpG island in the first intron and the first exon of the MCJ gene [7]. Loss of MCJ expression has been reported to correlate with chemoresistance of various tumors and poor survival of the patients as well as up-regulation of the ABCB1 drug transporter [8]. However, almost nothing was known about the physiological function of MCJ. The analysis of the cellular role of MCJ is a prerequisite to elucidate its contribution to the pathological processes. 

[Bildunterschrift / Subline]: Figure 1: Model of MCJ in the TIM23 translocase of human mitochondria. IMS: intermembrane space; IM: inner membrane

 

MCJ shows highest sequence homology to the Tim14 proteins, J protein family members which are named after E.coli DNAJ and act in conjunction with Hsp70 chaperone proteins [9]. By subcellular fractionation we demonstrated that endogenous MCJ is located in mitochondria. It is anchored in the mitochondrial inner membrane with its C-terminal J domain facing the matrix space. Using co-immunoprecipiation experiments, we showed that MCJ forms a stable subcomplex with a component of the mitochondrial import motor, MAGMAS, the human homologue of Tim16. MAGMAS has previously been shown to be overexpressed in cells treated with granulocyte-macrophage colony-stimulating factor and in prostate carcinomas [10]. In addition, MCJ and MAGMAS interact with the core components of the TIM23 translocase, human TIM17, TIM23 and TIM50. Employing an in vitro spectrophotometric assay we demonstrated that the recombinant soluble J domain of MCJ stimulates the ATPase activity of the human mtHsp70 chaperone, mortalin, the central component of the import motor of the TIM23 translocase. This stimulation is counteracted by MAGMAS. Moreover, we found that in vitro import of precursor proteins into mitochondria was impaired in the absence of MCJ, using a stable knock-down of MCJ in the MCF7 breast cancer cell line. Interestingly, MCJ was able to replace Tim14, the essential J co-chaperone of the mitochondrial protein import motor [11] [12], in yeast. In summary, our results showed that MCJ functions as a stimulating J co-chaperone of the human TIM23 preprotein translocase suggesting a link between mitochondrial preprotein import and tumorigenesis. With this characterization of MCJ as starting point, we aim to understand the molecular mechanisms of mitochondrial protein import and also to explain why two homologues evolved for distinct components of this vital process. To this end we investigate the components of the TIM23 translocase in different human cell lines. Studies of the recombinant proteins and complementation assays in the model organism S. cerevisiae will help us to obtain new information about the structure and function of the components of the human TIM23 translocase. The results of these analyses might also provide first insight into the origin and development of mitochondria related diseases.

Literature

1. Prokisch, H., et al., MitoP2: the mitochondrial proteome database--now including mouse data. Nucleic Acids Res., 2006. 34(Database issue): p. D705-D711.

2. Neupert, W. and J.M. Herrmann, Translocation of proteins into mitochondria. Annu. Rev. Biochem., 2007. 76: p. 723-749.

3. Mokranjac, D. and W. Neupert, The many faces of the mitochondrial TIM23 complex. Biochim. Biophys. Acta, 2010. 1797(6-7): p. 1045-1054.

4. Bauer, M.F., et al., Genetic and structural characterization of the human mitochondrial inner membrane translocase. J. Mol. Biol., 1999. 289(1): p. 69-82.

5. Schusdziarra, C., et al., Methylation-controlled J-protein MCJ acts in the import of proteins into human mitochondria. Hum. Mol. Genet., 2013. 22(7): p. 1348-1357.

6. Shridhar, V., et al., Loss of expression of a new member of the DNAJ protein family confers resistance to chemotherapeutic agents used in the treatment of ovarian cancer. Cancer Res., 2001. 61(10): p. 4258-4265.

7. Strathdee, G., et al., Cell type-specific methylation of an intronic CpG island controls expression of the MCJ gene. Carcinogenesis, 2004. 25(5): p. 693-701.

8. Hatle, K.M., et al., Methylation-controlled J protein promotes c-Jun degradation to prevent ABCB1 transporter expression. Mol. Cell Biol., 2007. 27(8): p. 2952-2966.

9. Fan, C.Y., S. Lee, and D.M. Cyr, Mechanisms for regulation of Hsp70 function by Hsp40. Cell Stress Chaperones, 2003. 8(4): p. 309-316.

10. Jubinsky, P.T., et al., Magmas expression in neoplastic human prostate. J. Mol. Histol., 2005. 36(1-2): p. 69-75.

11. Mokranjac, D., et al., Tim14, a novel key component of the import motor of the TIM23 protein translocase of mitochondria. EMBO J., 2003. 22(19): p. 4945-4956.

12. Truscott, K.N., et al., A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria. J. Cell Biol., 2003. 163(4): p. 707-713.


Scientific career
  • 2004-2009
  • Pharmaceutical studies at LMU, Munich
  • 05 - 10 / 2009
  • Internship at Bioproof AG, Munich: Quantitative analysis of small molecules and biologicals
  • since 2010
  • PhD student at LMU Munich, Prof. W. Neupert (since May 2011 Prof. A. Ladurner), lab of PD K. Hell "Structural and functional analysis of the TIM23 translocase of human mitochondria"

Publications
  • * Poster presentation at EMBO conference “From Structure to Function of Translocation Machines” April 2013
  • * Christina Schusdziarra, Marta Blamowska, Abdussalam Azem, Kai Hell: Methylation-controlled J-protein MCJ acts in the import of proteins into human mitochondria, Hum. Mol. Genet. 2012

Publications
  • * Marks D.S., Hopf T.A., Sander C. (2012). Protein structure prediction from sequence variation. Nature Biotechnology 30 (11), 1072-80.
  • * Hopf T.A., Colwell L. J., Sheridan R., Rost B., Sander C., Marks D. S. (2012). Three-dimensional structures of membrane proteins from genomic sequencing. Cell 149 (7), 1607-21.
  • * Hopf T.: Membrane Protein 3D Structure from Sequence Alone (2012). Master's Thesis, Technische Universität München.