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Ultrastructural analysis of yeast cell mitochondria

von Ann-Katrin Unger

The aim of this thesis is to identify new components (proteins) that are involved in maintaining mitochondrial ultrastructure. The unicellular eukaryote Saccharomyces cerevisiae (budding yeast) is a very good model organism to solve these questions. The benefits are for example a fully sequenced genome which makes it easy to genetically manipulate the organism.

Mitochondria are dynamic organelles that constantly fuse and divide [1]. They are the powerhouses of the cell and responsible for oxidative phosphorylation. They play an important role in many fundamental cellular processes like apoptosis, aging, metabolism, reactive oxygen species detoxification and ATP production [2]. Their function is directly related to their architecture. Dysfunction of the organelle can lead to human diseases. Although mitochondrial architecture differs from cell types to tissues, a basic common structure is maintained. Even though the functions of mitochondria are analyzed in detail, the origin and maintenance of this defined architecture remains unclear.

During the recent years I worked on the ultrastructural analysis of mitochondria in the yeast Saccharomyces cerevisiae. The aim of my thesis is to identify new components (proteins) that are involved in maintaining mitochondrial ultrastructure. The unicellular eukaryote Saccharomyces cerevisiae (budding yeast) is a very good model organism to solve these questions. The benefits are for example a fully sequenced genome which makes it easy to genetically manipulate the organism. The cells have a short generation time of 1-2 h and importantly they are able to switch to fermentation if energy consumption via mitochondria cannot take place. In contrast to other eukaryotic model organisms, the yeast is able to survive without respirational active mitochondria.

To analyze mitochondrial alterations on the morphological level I made use of microscopic techniques like fluorescent and transmission electron microscopy (TEM). The TEM analysis makes it possible to get detailed insights into the organelles architecture because a much higher resolution can be achieved compared to light microscopy. Protein localizations can be determined via Immunogold labeling and 3D impressions of the cell can be achieved via electron tomography. The mitochondrial network morphology can be analyzed in living cells via fluorescence microscopy.

Mitochondria are surrounded by two membranes: the outer mitochondrial membrane (MOM) and the inner mitochondrial membrane (MIM). The outer mitochondrial membrane forms a barrier to the cytosol and is necessary for the exchange of proteins and lipids with the surrounding cytosol [3]. The inner mitochondrial membrane can be subdivided into a) the inner boundary membrane, which is in close proximity to the outer mitochondrial membrane and b) the cristae membranes, which are invaginations into the mitochondrial matrix space and which are the sites of oxidative phosphorylation. At the sites where those invaginations arise the so called cristae junctions (CJs) are located. They are necessary to form contact sites to the mitochondrial outer membrane to enable protein import into mitochondria [3]. Recent studies have identified a considerable number of proteins responsible for influencing mitochondrial architecture. An important protein complex, the MICOS complex (mitochondrial contact site and cristae organizing system), consisting out of six subunits (Mic10, Mic12, Mic19, Mic26 Mic27 and Mic60) was identified to be responsible for the formation and maintenance of cristae junctions (CJ) in yeast [4,5,6].
In a previously reported proteomics screen for proteins which accumulate at mitochondrial contact sites a protein exhibiting the same distribution as the subunits of the MICOS complex was identified [4]. This protein of unknown function is localized to mitochondria and is the product of the AIM24 gene, which had been found in a previous screen for mutants with altered inheritance of mitochondria [7, 8]. We expected that mitochondrial ultrastructure is altered in the absence of Aim24 because deletion of Aim24 affected the integrity of the MICOS complex. Therefor we performed an electron microscopic analysis of cells lacking Aim24. We found that the loss of Aim24 leads to an increase of inner membrane septa and to abnormal mitochondrial morphology (Figure 2). The morphological defect is enhanced when cells are grown under respiratory conditions and leads to a loss of correct respiratory function of the mitochondria [9].

The architectural role of Aim24 is probably caused by the interaction with the MICOS complex, which is necessary to form cristae junctions. The direct role of Aim24 in this complex network of regulating and forming mitochondrial architecture is probably in stabilizing the MICOS complex but further research has to be performed to characterize the function in more detail [9].

[1] Westermann, B. (2010). "Mitochondrial fusion and fission in cell life and death." Nature Rev. Mol. Cell Biol. 11: 872-844.

[2] Cogliati, S., J. A. Enriquez and L. Scorrano (2016). "Mitochondrial Cristae: Where Beauty Meets Functionality." Trends Biochem Sci.

[3] van der Laan, M., S. E. Horvath and N. Pfanner (2016). "Mitochondrial contact site and cristae organizing system." Curr Opin Cell Biol 41: 33-42.

[4] Harner, M., C. Korner, D. Walther, D. Mokranjac, J. Kaesmacher, U. Welsch, J. Griffith, M. Mann, F. Reggiori and W. Neupert (2011). "The mitochondrial contact site complex, a determinant of mitochondrial architecture." EMBO J 30(21): 4356-4370.

[5] Hoppins, S., S. R. Collins, A. Cassidy-Stone, E. Hummel, R. M. Devay, L. L. Lackner, B. Westermann, M. Schuldiner, J. S. Weissman and J. Nunnari (2011). "A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria." J Cell Biol 195(2): 323-340.

[6] von der Malsburg, K., J. M. Muller, M. Bohnert, S. Oeljeklaus, P. Kwiatkowska, T. Becker, A. Loniewska-Lwowska, S. Wiese, S. Rao, D. Milenkovic, D. P. Hutu, R. M. Zerbes, A. Schulze-Specking, H. E. Meyer, J. C. Martinou, S. Rospert, P. Rehling, C. Meisinger, M. Veenhuis, B. Warscheid, I. J. van der Klei, N. Pfanner, A. Chacinska and M. van der Laan (2011). "Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis." Dev Cell 21(4): 694-707.

[7] Hess DC, Myers CL, Huttenhower C, Hibbs MA, Hayes AP, Paw J, Clore JJ, Mendoza RM, Luis BS, Nislow C, Giaever G, Costanzo M, Troyanskaya OG, Caudy AA. 2009. Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis. PLOS Genetics 5:e1000407.
[8] Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O’Shea EK. 2003. Global analysis of protein localization in budding yeast. Nature 425:686–691.

[9] Harner, M. E., A. K. Unger, T. Izawa, D. M. Walther, C. Ozbalci, S. Geimer, F. Reggiori, B. Brugger, M. Mann, B. Westermann and W. Neupert (2014). "Aim24 and MICOS modulate respiratory function, tafazzin-related cardiolipin modification and mitochondrial architecture." Elife 3: e01684.

Zur Person

Ann-Katrin Unger absolvierte 2011 ihr Bachelor-Studium der Biologie an der Universität Bayreuth. Im Anschluss studierte sie bis 2013 den Studiengang Molekulare Ökologie, welchen sie mit dem akademischen Grad Master of Science erfolgreich abschloss. Seit 2013 promoviert sie am Institut für Zellbiologie und erhält ein Promotionsstipendium der Max-Planck Gesellschaft. (23.09.2016)


Figure 1: Schematic overview of (A) mitochondrial architecture (B) the localization of the MICOS (mitochondrial contact site and cristae organizing system) complex and respiratory chain complexes (OXPHOS) at the cristae membranes. Modified after van der Laan et al. 2016 [3].

Figure 2: Electron micrographs of (A) wild type mitochondria and (B) altered mitochondria (lacking the Aim24 protein) of Saccharomyces cerevisiae, grown on respiratory carbon source. Scale bars: 2 µm

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