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. 2014 Jun 19:5:4129.
doi: 10.1038/ncomms5129.

Visualizing active membrane protein complexes by electron cryotomography

Vicki A M Gold  1 , Raffaele Ieva  2 , Andreas Walter  1 , Nikolaus Pfanner  2 , Martin van der Laan  2 , Werner Kühlbrandt  1
Affiliations
Free PMC article

Visualizing active membrane protein complexes by electron cryotomography

Vicki A M Gold et al. Nat Commun. .
Free PMC article

Abstract

Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future.

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Figures

Figure 1
Figure 1. Design and implementation of the approach.
(a) Schematic of the STAMP (Specifically TArgeted Membrane nanoParticle) approach. In the first step, a small biotin label (yellow) is attached via a spacer arm (orange) to a mitochondrial model preprotein (red). Subsequently, the labelled preprotein is targeted to the mitochondrial import machinery and arrested by means of a tightly folded DHFR domain as a translocation intermediate spanning both TOM (blue) and TIM23 (cyan) complexes. In the third step, the DHFR-linked biotin is bound by streptavidin (purple)-conjugated QDs (black sphere with green spacer arms). The total distance from the QD to the outer mitochondrial membrane is ~10 nm. (b) The design of the mitochondrial model preprotein is based on the fusion protein b2Δ-DHFR. To enable site-specific biotinylation, endogenous cysteines (C14, C86 and C157) were substituted with serine and a unique cysteine replaced the C-terminal residue D337. (c) Blue native electrophoresis and western blot analysis of the preprotein-tethered TOM–TIM23 supercomplex detected with an antibody against Tim23. Note that the TIM23 core complexes (TIM23CORE) are quantitatively shifted into TOM–TIM23 supercomplexes on addition of b2Δ-DHFRbiotin, indicating that the import sites are occupied by preproteins under these conditions. The experiment was repeated three times. (d) Free QDs were separated from labelled mitochondria on an OptiPrep gradient. Under white light (tubes 1 and 3) mitochondrial membranes are visible (M, yellow boxes), and under UV excitation, QD525 is detected (tubes 2 and 4; green boxes). QD525 co-localization with mitochondria is seen in tube 4.
Figure 2
Figure 2. Preprotein import sites are non-uniform.
(a) A slice through a tomogram reveals the location of QDs (green arrowheads); scale bar, 100 nm, inset=10 nm. (b) Segmentation of the volume shows the distribution of QDs (green spheres) around the mitochondrion. The outer membrane (purple), the inner membrane (lilac) and the crista membranes (cyan) are also shown. (c) Close-ups of the import sites reveal the position of clusters relative to mitochondrial membranes and CJs (yellow arrowheads).
Figure 3
Figure 3. Specific labelling determines the total number of preprotein import sites and the degree of clustering.
(a) Free QDs were separated from labelled mitochondria on an OptiPrep gradient. Under white light (tubes 1 and 3) mitochondria are visible (M, yellow boxes), and under UV excitation, QD605 is detected (tubes 2 and 4; green boxes). QD605 co-localization with mitochondria is seen in tube 4. The experiment was repeated four times. (b) Confocal fluorescence images of non-importing and importing mitochondria. The mitochondria are labelled with MitoTracker Green and QD605 fluorescence is shown in red; scale bar, 1 μm. The black space between mitochondria has been removed (original in Supplementary Fig. 5a). The statistics described in the text were calculated based on n=350 for the control and n=140 for the actively importing mitochondria. (c) A slice near the top of the tomogram reveals the location of a cluster of QDs (green arrowheads) on the mitochondrial surface. A slice through the centre shows the position of the cluster with respect to the CJs (yellow arrowheads); scale bar, 100 nm, cluster measures ~80 × 60 nm. (d) Segmentation of the volume depicts the three-dimensional distribution of QDs (green spheres) around the mitochondrion relative to the outer membrane (blue) and the cristae (yellow). (e) A model of an ellipsoid was used to calculate the surface area of mitochondria based on a, b and c radii as depicted. (f) A line graph shows the correlation between mitochondrial size and the number of preprotein import sites, n=12 mitochondria as seen in Table 2. (g) Averaged histogram showing the closest distance between two QDs, calculated from 12 mitochondrial samples (Supplementary Fig. 6) accumulating 1,159 QD605 data points in total. Error bars indicate the s.d. of the frequency distribution for each minimal distance.
Figure 4
Figure 4. Preprotein import occurs at discrete microdomains.
(a) A slice near the top of a tomogram reveals the location of clusters of QDs (green arrowheads) on the mitochondrial surface. A slice through the centre shows further QDs and the fusion septum (purple arrowhead); scale bar, 100 nm, cluster measures ~150 × 60 nm. Segmentation of the volume depicts the three-dimensional distribution of QDs (green spheres) around the mitochondrion with respect to the cristae (yellow) and around the mitochondrial septum. The outer membrane is rendered in two colours (light and dark blue) in order to distinguish the position of the septal ring. (b) Partial segmentation of the upper surface of a mitochondrion reveals the position of QDs (green spheres) with respect to a slit-like CJ (yellow) on the inner boundary membrane (mauve). For clarity, the outer membrane is not shown; scale bar, 100 nm, cluster measures ~50 × 250 nm. (c) Averaged histogram showing the closest distance between QD and CJs, calculated from nine mitochondrial samples (Supplementary Fig. 8) accumulating 836 QD605 data points in total. Error bars indicate the s.d. of the frequency distribution for each minimal distance.
Figure 5
Figure 5. Details of the import supercomplex.
Left panels: close inspection of the protein densities localized 10 nm beneath the QDs (green arrowheads) reveals characteristic features on the outer membrane (blue arrowheads) and protein densities spanning the intermembrane space (red arrowheads). The multiple panels in a and b represent different slices through the same tomogram; in c all features were visible in one slice; scale bars, 10 nm. Right panels: the segmented volumes of the same features in three dimensions (QDs green).
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