Global, quantitative and dynamic mapping of protein subcellular localization
- Max Planck Institute of Biochemistry, Germany
Figures
Figure 1 with 1 supplement
Generation of organellar maps through fractionation profiling.
(A) Metabolically labelled HeLa cells were mechanically lysed to release organelles. Light labelled lysate was then subjected to differential centrifugation at the indicated speeds (RCFMAX) and …
Figure 1—figure supplement 1
Organellar leakage analysis (A) and fractionation reproducibility (B, C).
(A) Leakage of lumenal contents from endoplasmic reticulum, mitochondria and lysosomes was calculated by quantifying the cytosolic pool of lumenal marker proteins (see Figure 1C, and Materials and me…
Figure 2 with 2 supplements
Visualization of an organellar map.
Thirty SILAC ratios from six replicate fractionation experiments were combined and subjected to principal component analysis to achieve dimensionality reduction. Projections along the first (x-axis) …
Figure 2—figure supplement 1
Organellar map with full cluster annotation (A); overlay of an organellar map with external protein sequence feature predictions (B).
(A) Close inspection of the map shown in Figure 2 reveals sub-clustering within the main clusters. The mitochondrial cluster shows separation into outer membrane, inner membrane and matrix proteins; …
Figure 2—figure supplement 2
Reproducibility analysis of organellar maps.
(A) Six individual maps (with five SILAC ratios each) were visualized by PCA as described in Figure 2. Maps were made in pairs (1&2, 3&4, 5&6), on three separate days. Notice the highly reproducible …
Figure 3
Quantitative anatomy of a HeLa cell.
(A) Schematic diagram of a cell where compartments are approximately scaled by their relative contributions to total cell protein mass (not by their volumes). All membranous organelles combined …
Figure 4 with 3 supplements
Dynamic organellar maps reveal protein localization changes following EGF stimulation.
(A, B) Fluorescently tagged EGF (green) was pre-bound to HeLa cells on ice, and imaged by confocal microscopy. Lysosomal compartments were visualized with Lysotracker (red). Most of the EGF was at …
Figure 4—figure supplement 1
Dynamic organellar maps (EGF-treatment) – overview of the experimental workflow.
Starting with SILAC light and heavy cells in both conditions, lyse each batch of cells separately. Subject the lysates to differential centrifugation, generating membrane sub-fractions with light …
Figure 4—figure supplement 2
Protein localization changes following EGF stimulation.
(A) Organellar maps were prepared from untreatedHeLa cells (control, left side), and from cells following continuous stimulation with EGF for 20 min (+EGF, right side). The individual maps from …
Figure 4—figure supplement 3
Global protein distribution profile changes induced by EGF treatment.
For proteins undergoing significant localization changes (Figure 4—figure supplement 1, Supplementary file 7), the distribution between nuclear, organellar and cytosolic fractions is shown before …
Figure 5
Quantitative mapping of EGF-triggered subcellular translocation events.
Summary of key protein translocations in HeLa cells following 20 min of continuous stimulation with EGF. All depicted changes were detected by organellar maps in this study; they include numerous …
Tables
Table 1
Prediction output and performance of HeLa organellar maps. The table shows the combined organellar prediction output from six replicate maps from HeLa cells. Prediction performance is judged by the …
| Compartment | Number of marker proteins | Correctly predicted markers | All proteins predicted in this compartment | |
|---|---|---|---|---|
| Number | % | |||
| Endosome | 85 | 75 | 88.2% | 304 |
| ER | 127 | 127 | 100.0% | 530 |
| ER, high curvature | 11 | 11 | 100.0% | 45 |
| ERGIC/cisGolgi | 26 | 25 | 96.2% | 73 |
| Golgi | 33 | 29 | 87.9% | 190 |
| Lysosome | 43 | 41 | 95.3% | 88 |
| Mitochondrion | 242 | 239 | 98.8% | 658 |
| Peroxisome | 21 | 15 | 71.4% | 25 |
| Plasma membrane | 127 | 123 | 96.9% | 510 |
| All organellar proteins | 715 | 685 | 95.8% | 2423 |
| Average per organelle | 92.7% | |||
| Large Protein Complexes | 361 | 353 | 97.8% | 2739 |
| Total | 1076 | 1038 | 96.5% | 5162 |
Additional files
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Supplementary file 1
The HeLa spatial proteome.
A compact summary of organellar assignments and abundance information. Also includes the organellar markers used for classification.
- https://doi.org/10.7554/eLife.16950.015
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Supplementary file 2
Prediction performance.
The performance and depth of all 12 maps reported in this study.
- https://doi.org/10.7554/eLife.16950.016
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Supplementary file 3
External validation of predictions.
Contains the concordance analysis with external protein subcellular localization information. Our predictions are compared to UniProt annotations, and to a mouse cell line spatial proteome.
- https://doi.org/10.7554/eLife.16950.017
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Supplementary file 4
The complete HeLa protein subcellular localization database.
An interactive database containing the full spatial information generated in this study, including localization, copy numbers, and neighbourhood analysis.
- https://doi.org/10.7554/eLife.16950.018
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Supplementary file 5
Anatomy of major organelles.
The quantitative composition of three major compartments, ER, mitochondria, and plasma membrane.
- https://doi.org/10.7554/eLife.16950.019
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Supplementary file 6
EGF dynamics database.
An interactive database showing organellar predictions and abundance changes induced by EGF.
- https://doi.org/10.7554/eLife.16950.020
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Supplementary file 7
EGF Translocation analysis.
A summary of detected organellar and nuclear/cytosolic translocation events triggered by EGF.
- https://doi.org/10.7554/eLife.16950.021
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Supplementary file 8
Comparison of organellar profiling approaches.
An overview of the features and requirements of Dynamic Organellar Maps and the LOPIT approach.
- https://doi.org/10.7554/eLife.16950.022
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Supplementary file 9
Raw data.
The quantitative proteomics data used in this study (SILAC, intensity and LFQ data).
- https://doi.org/10.7554/eLife.16950.023
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Supplementary file 10
How to use www.MapOfTheCell.org.
- https://doi.org/10.7554/eLife.16950.024
