As the primary cellular source of ATP, mitochondria form a vital bioenergetic, metabolic and signaling hub. Despite the presence of mitochondrial DNA, almost all mitochondrial proteins (99%) are nuclear encoded and are imported from the cytosol. Protein translocases in mitochondrial membranes are therefore essential for correct protein targeting and localisation. Mitochondrial dysfunction is implicated in ageing, as well as a growing number of human pathologies, including certain cancers, genetically inherited syndromes and neurodegenerative disorders such as Alzheimer’s disease. The biogenesis of mitochondrial proteins and import sites are therefore important factors that determine organelle functionality.

The broad aim of this research is to understand how protein import is correctly orchestrated and regulated by innovative approaches focussed on state-of-the-art electron cryo-tomography (cryoET). This technique is the method of choice for the study of proteins or complexes in situ (Figure 1). Samples are preserved by cryo-fixation, imaged in the electron microscope, and protein structures can be determined by subtomogram averaging (StA).

The majority of precursor proteins (preproteins) are actively imported into mitochondria from the cytosol through the Translocase of the Outer Membrane (TOM) complex, which forms a common entry gate for proteins that are subsequently targeted to various locations. For decades, it has been known that proteins can be imported into mitochondria post-translationally, after synthesis on ribosomes in the cytosol or in vitro. However, information regarding the organisation and distribution of mitochondrial protein import sites was sparse. To image active post-translational protein import in situ, we previously devised a new labelling strategy in which mitochondrial-targeted preproteins were arrested as two-membrane-spanning intermediates through both the TOM complex and the Translocase of the Inner Membrane (TIM23) complex concurrently (Gold, V. A. M. et al. (2014) Nat Commun 5, 4129). This work provided the first views of mitochondrial proteins in the act of import and enabled direct visualization of the number and location of active complexes for the first time (Figure 1). However, information regarding the cytosolic stage of mitochondrial precursor protein import and targeting was still lacking.

Most recently, we have developed a new method to investigate the cytosolic stage of mitochondrial protein targeting and import by biochemical techniques, cryo-ET and StA.  This reveals unprecedented details regarding different targeting pathways, shedding light on the interaction between importing proteins and their corresponding membrane-bound translocons. The consequences of these different modes of targeting and import site redistribution will be discussed in the context of mitochondrial protein biogenesis.

Legend to Figure 1

1. Top panel: mitochondria embedded in a layer of amorphous ice are imaged in the electron microscope.  Incremental tilts of the sample yield a series of projections from different viewing angles. The relative orientations of the mitochondrion (brown) and the macromolecular complex of interest (red) vary depending on the projection angle. Bottom panel: a 0° tilt projection image of a S. cerevisiae mitochondrion.

2. Top panel: a three-dimensional tomogram is reconstructed from the two-dimensional image series by computational back-projection. The molecular complex of interest is indicated (red, boxed). Bottom panel: a slice through a reconstructed tomogram of the mitochondrion shown in step 1. Labelled importing proteins (black spheres on the outer membrane are indicated (red arrowhead).

3. Top left and bottom panels: surface volume rendering is used to place complexes of interest back into three-dimensional space in order to visualize their distribution in a native-like context. Labelled preproteins (black spheres) are shown on the mitochondrial outer membrane (green). The inner membrane (blue) and crista membranes (yellow) are also shown. Top right panel: a slice through the reconstructed tomogram of a labelled preprotein (black sphere, red arrowhead) engaged in mitochondrial import.

Figures:

Figure 1. Workflow for cryoET of mitochondria, actively engaged in preprotein import. _x000D_Acknowledgements: Martin van der Laan, Raffaele Ieva, Nikolaus Pfanner, Misha Kudrayashev, Bertram Daum, Deryck Mills, Werner Kühlbrandt

« Back to The 16th European Microscopy Congress 2016

EMC Abstracts - https://emc-proceedings.com/abstract/insights-into-mitochondrial-protein-import-studied-by-cryoet/