Determination of the supramolecular organisation of mitochondrial import sites in situ by electron cryotomography
Mitochondria are the essential energy-providing organelles of the eukaryotic cell. During evolution, the mitochondrial genome became degenerate and as such, 99% of proteins are now nuclear encoded. These must subsequently overcome a major obstacle, that is transport from the cytosol across or into a hydrophobic lipid bilayer.
Protein translocases in the membranes are required for protein targeting and correct sub-cellular localisation. Such assemblies form large and complex structures composed of soluble and membrane-anchored subunits, working together to orchestrate the import of preproteins to their required destination.
We focus on the transport of matrix targeted proteins through the TOM and TIM23 complexes in the outer and inner mitochondrial membranes respectively. A preprotein with the ability to arrest during translocation may be used as a tool to enrich for such TOM-TIM23 supercomplexes in organello (Figure 1). We are investigating different ways of tagging the protein in order to render it dense in the electron microscope, to facilitate supercomplex localisation by electron cryotomography (Gold et al, Nat. Commun. 2014).
Figure 1: Pre-proteins destined for the matrix contain N-terminal pre-sequences, which target them to the TOM complex in the outer membrane via the Tom22 receptor. Due to ever increasing binding affinities, the substrate is then passed via Tom22 and Tom5 to the Tom40 channel. As the pre-protein emerges on the intermembrane space side, it is passed to the TIM23 complex in the inner membrane. Full translocation into the matrix is dependent on the ΔΨ and the import motor ATPase PAM. A pre-protein with a C-terminal tightly folded domain can be arrested across both membranes concurrently, bridging the TOM and TIM23 complexes together. Visualisation of such structures in the electron microscope may be assisted by inclusion of dense tags on the C-terminus of the import substrate.
Electron cryotomography (cryoET) is a powerful tool, which we can use to visualise mitochondria in a native-like environment. A series of tilted projections are used to create a three-dimensional volume of a vitrified sample.
Figure 2: A series of images is collected from -/+ ~60° in the electron microscope. The tilt series is then reconstructed to build a tomographic volume of the original sample. The tomogram may then be segmented in different colours to assist visualisation in three dimensions.
We combine preprotein labelling of the import supercomplex with tomography of isolated yeast mitochondria, in order to reveal fascinating details of the import machinery at work.
Figure 3: Biotinylated preproteins en route to the mitochondrial matrix are labelled via streptavidin conjugated quantum dots (black spheres). The non-uniform distribution of import sites and clustering near crista junctions can be demonstrated using cryoET (red box; cristae are shown in yellow and the mitochondrial outer and inner membranes in green and blue respectively). This technique may also be used to reveal details of the import machinery itself (blue box; a black arrowhead points to the quantum dot, blue and green arrowheads point to densities protruding from the outer membrane and crossing the intermembrane space respectively. For further details see Gold et al, Nat. Commun. 2014.
Vicki A. M. Gold#, Ralf Salzer, Beate Averhoff and Werner Kühlbrandt (2015). Structure of a type IV pilus machinery in the open and closed state. eLife. (10.7554/eLife.07380).
# corresponding author
Vicki A. M. Gold#, Raffaele Ieva, Andreas Walter, Martin van der Laan, Nikolaus Pfanner & Werner Kühlbrandt (2014). Visualizing active membrane complexes by electron cryotomography. Nat. Commun. (10.1038/ncomms5129).
# corresponding author
Karen M. Davies, Bertram Daum, Vicki A. M. Gold, Alexander W. Mühleip, Tobias Brandt, Thorsten B. Blum, Deryck J. Mills & Werner Kühlbrandt (2014). Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography. JoVE (doi: 10.3791/51228).
Carola Stockburger, Vicki A. M. Gold, Thea Pallas, Natalie Kolesova, Davide Miano, Kristina Leuner & Walter E. Müller (2014). A cell model for the initial phase of sporadic Alzheimer’s disease. J. Alz. Dis. 42(2):395-411.
Ryan J. Schulze, Joanna Komar, Mathieu Botte, Sarah Whitehouse, Vicki A. M. Gold, Karine Huard, Imre Berger, Christiane Schaffitzel & Ian Collinson (2014). Membrane protein insertion and PMF-driven protein export by the bacterial holo-translocon SecYEG-SecDF-YajC-YidC. PNAS 111(13): 4844-4849.
Vicki A. M. Gold, Sarah Whitehouse, Alice Robson & Ian Collinson. (2013). The dynamic action of SecA during the initiation of protein translocation. Biochem J. 1;449(3):695-705.
Sarah Whitehouse, Vicki A. M. Gold, Richard B. Sessions & Ian Collinson. (2012). Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex. JCB. 199(6):919-29.
Dilem Hizlan, Alice Robson, Sarah Whitehouse, Vicki A.M. Gold, Janet Vonck, Deryck Mills, Werner Kühlbrandt & Ian Collinson (2012). Structure of the SecY complex unlocked by a preprotein mimic. Cell Reports. 1;449(3):695-705.
Karine Deville, Vicki A.M. Gold*, Alice Robson, Richard B. Sessions, Stephen Baldwin, Sheena Radford & Ian Collinson (2011). The oligomeric state and arrangement of the active bacterial translocon. JBC. 286(6):4659–69. *Equal first author contribution
Vicki A.M. Gold, Alice Robson, Huan Bao, Tatyana Romantsov, Franck Duong & Ian Collinson (2010). The action of cardiolipin on the bacterial translocon. PNAS. 107(22):10044-9.
Alice Robson, Vicki A.M. Gold*, Skye Hodson, Anthony R. Clarke and Ian Collinson. (2009). Energy transduction in protein transport and the ATP hydrolytic cycle of SecA. PNAS. 106(13) 5111-5116. *Equal first author contribution
Vicki A.M. Gold, Alice Robson, Anthony R. Clarke and Ian Collinson. (2007). Allosteric regulation of SecA: magnesium-mediated control of conformation and activity. J Biol Chem. 282(24) 17424-32.
Vicki A.M. Gold, Franck Duong and Ian Collinson. (2007). The bacterial protein translocation reaction driven through the SecY channel by the ATPase SecA. Mol Membr Biol. 24(5-6): 387-94. Review.
Alice Robson, Antonia E. Booth, Vicki A.M. Gold, Anthony R. Clarke and Ian Collinson (2007). A Large Conformational Change Couples the ATP Binding Site of SecA to the SecY Protein Channel. J Mol Biol. 374(4) 965-76.
Biochemistry tutor and laboratory demonstrator, 2004-2007
Meetings and Courses
- GRC: Three Dimensional Electron Microscopy 2013, New England, USA (poster)
- EMBO Fellows Meeting 2013, Heidelberg, Germany (talk)
- EMBO Meeting: From Structure to Function of Translocation Machines 2013, Dubrovnik, Croatia (talk)
- The EMBO Meeting 2012. Nice, France (poster)
- European Bioenergetics Conference 2012, Freiburg, Germany (talk)
- GBM Molecular Life Sciences meeting 2011, Frankfurt, Germany
- EMBO Meeting: Protein Transport Systems 2011, Santa Margherita di Pula, Sardegna, Italy (poster)
- GRC: Protein Transport Across Cell Membranes 2010, Galveston, Texas, USA (poster)
- EMBO PostDoc Laboratory Management Course 2009, Heidelberg, Germany
- International FOR 967 Meeting 2009, Homburg, Germany
- SGM Meeting: Protein targeting 2008. Edinburgh, Scotland (poster)
- Molecular Chaperone Club 18th Annual Meeting 2008. Bristol, UK
- FEBS-EMBO Summer School: Protein, Lipid and Membrane Traffic: Pathways and Targeting 2007, Cargese, Corsica (poster)
- ASM-FEMS Meeting: Bacterial protein trafficking 2006, Crete, Greece (poster)
- Max-Planck-Institute International Symposium
– Functional Membrane Proteomics 2005, Frankfurt, Germany
- Biochemical Society, Bioscience 2005: From genes to systems, Glasgow, Scotland (poster)
- Cellular Protein Translocation: Experiment and Theory 2005, University of Warwick, UK
Dr. Vicki Gold
Department of Structural Biology
Phone: +49 (0) 69 6303-3050
Fax: +49 (0) 69 6303-3002
Research Groups and Project Leaders
Ph. D. Students