The organelle proteome

Spatial compartmentalization of biological functions is a fundamental strategy that enables multiple biological processes to occur in parallel without undesired interference. An organelle is a subunit of the eukaryotic cell with a specialized function. The name "organelle" stems from the analogy between the different roles of organelles in the cells to the different roles of organs in the human body as a whole. A distinction is often made between membrane-bound and non-membrane bound organelles. The membrane-bound organelles, such as the nucleus and the Golgi apparatus, have a clearly defined physical boundary that separates the internal space from the outside. In contrast, non-membrane bound organelles and subcellylar structures, like the cytoskeleton and nucleoli, constitute spatially distinct assemblies of proteins, and sometimes RNA, within the cell without a physical boundary. In either case, this partitioning of cellular components creates specific environments where the concentration of different molecules can be tailored to fit the purpose of the organelle or subcellular structure, and provides important opportunities for regulation and coordination of cellular processes.

A major function of proteins is to catalyze, conduct and control cellular processes in time and space. As different organelles and subcellular structures offer distinct environments, with distinct physiological conditions and interaction partners, the subcellular localization of a protein is an important part of protein function. Consequently, mis-localization of proteins have often been associated with cellular dysfunction and various human diseases (Kau TR et al. (2004); Laurila K et al. (2009); Park S et al. (2011)). Knowledge of the spatial distribution of proteins at the subcellular level is essential for understanding protein functions and protein interactions, as well as identifying the components of different cellular processes. Thus, studying how cells generate and maintain their spatial organization is central for understanding the functions and mechanisms of living cells.

Within the Subcellular Section, 13041 human proteins have been mapped on a single-cell level to 35 different organelles and subcellular structures (Figure 1), which has enabled the definition of 13 major organelle proteomes:

The analysis also reveals that approximately half of the proteins localize to multiple compartments and identifies many proteins with single-cell variation in terms of protein abundance and/or spatial distribution.

Subcellular localization of proteins

Several approaches for systematic analysis of protein localization have been described. Quantitative mass-spectrometric readouts allow identification of proteins with similar distribution profiles across fractionation gradients (Park S et al. (2011); Christoforou A et al. (2016); Itzhak DN et al. (2016)) or enzyme-mediated proximity-labelled proteins in cells (Itzhak DN et al. (2016); Roux KJ et al. (2012); Lee SY et al. (2016)). In contrast, imaging-based approaches enable the exploration of subcellular distribution of proteins in situ in single cells and have the advantage of effectively identifying single-cell variability and multi-organelle localization. Imaging based approaches can be performed using affinty tagged or fluorescently tagged recombinant proteins (Huh WK et al. (2003); Simpson JC et al. (2000); Stadler C et al. (2013)) or affinity reagents.

The Subcellular Section employs an immunofluorescence (IF) based approach combined with confocal microscopy to enable high-resolution investigation of the spatial distribution of proteins (Thul PJ et al. (2017); Stadler C et al. (2013); Barbe L et al. (2008); Stadler C et al. (2010); Fagerberg L et al. (2011)). With the diffraction-limited resolution of about 200 nm, an confocal image gives detailed insights into organization at the subcullar level. The spatial distribution of the protein is investigated using indirect IF in up to three cell lines, usually including U-2 OS and two additional cell lines selected based on mRNA expression of the corresponding gene, using a subset of 36 of the cell lines found in the Cell Line Section of the Human Protein Atlas. The protein of interest is visualized in green, while reference markers for microtubules (red), endoplasmic reticulum (yellow) and nucleus (blue) are used to outline the cell. From small dots like nuclear bodies, to larger structures such as the nucleoplasm, the distinct patterns in the images together with the reference markers make it possible to precisely determine the spatial distribution of a protein within the cell. The localization of each protein is assigned to one or more of 35 organelles and subcellular structures (Figure 1).


Nucleoplasm

Nuclear speckles

Nuclear bodies

Nucleoli

Nucleoli fibrillar center

Nucleoli rim

Mitotic chromosome

Kinetochore

Nuclear membrane

Cytosol

Cytoplasmic bodies

Rods & Rings

Aggresome

Mitochondria

Centrosome

Centriolar satellites

Microtubules

Microtubule ends

Mitotic spindle

Cytokinetic bridge

Midbody

Midbody ring

Cleavage furrow

Intermediate filaments

Actin filaments

Focal adhesion sites

Endoplasmic reticulum

Golgi apparatus

Vesicles

Endosomes

Lysosomes

Lipid droplets

Peroxisomes

Plasma membrane

Cell junctions

Figure 1. An example of confocal immunofluorescence images of different proteins (green) localized to each of the subcellular organelles and substructures currently annotated in the Subcellular Section in a representative set of cell lines. Microtubules are marked with an anti-tubulin antibody (red) and the nucleus is counterstained with DAPI (blue). For more example images and details describing all the 35 patterns annotated in the Subcellular Section of the Atlas, see the Cell Dictionary.

Protein distribution in human cells

Figure 2 shows the distribution of all classificactions across the 35 organelles and subcellular structures for 13041 genes with protein localization data in the Subcellular Section. The plot is sorted by meta-compartments: cytoplasm, nucleus, and secretory machinery, respectively. Most proteins are found in the nucleus, followed by the cytosol and vesicles, which consist of transport vesicles as well as small membrane-bound organelles like endosomes or peroxisomes. 56% (n=7329) of the proteins were detected in more than one location (multilocalizing proteins), and 24% (n=3193) displayed single-cell variation in expression level or spatial distribution.

Figure 2. Bar plot showing the distribution of classifications of proteins in organelles and subcellular structures in the Subcellular Section. Note that one protein can localize to more than one compartment. The bars are colored according to meta

Validation of antibodies and location data for the Subcellular Section

The quality and use of antibodies in research have been frequently debated (Baker M. (2015)). As antibody off-target binding can cause false positive results, the Subcellular section makes an effort in manually scoring all results regarding reliability of the staining. In the Subcellular Section a reliability score for every annotated location at a four-graded scale is provided: Enhanced, Supported, Approved, and Uncertain, as described in detail in the assay & annotation section. The enhanced locations are obtained through antibody validation according to one of the validation "pillars" proposed by an international working group (Uhlen M et al. (2016): (i) genetic methods using siRNA silencing (Stadler C et al. (2012)) or CRISPR/Cas9 knock-out, (ii) expression of a fluorescent protein-tagged protein at endogenous levels (Skogs M et al. (2017)) or (iii) independent antibodies targeting different epitopes (Stadler C et al. (2010)). A supportive location is in agreement with external experimental data (UniProt database), while an approved location score indicates that there is no external experimental information available to confirm the observed location. An uncertain location is contradictory compared to complementary information, such as literature or transcriptomics data, and is shown if it cannot be ruled out that the data is correct, and further experiments are needed to establish the reliability of the antibody staining. The distribution of reliability scores for the localized proteins is shown in Figure 3. Approximately 43% (n=5574) of the protein localizations provided are enhanced or supported. Table 1 details the organelle distribution of all localized proteins and the distribution of reliability scores on the basis of the individual organelle.

Figure 3. Pie chart showing level of reliability of the localized proteins, where each piece is the number of proteins with one type of score, out of the four reliability scores Enhanced, Supported, Approved, and Uncertain.

Table 1. Table showing the number of proteins localized to every organelle, structure, and substructure in the Subcellular Section, along with the distribution of reliability scores.

Location Proteins Location reliability
% Enhanced Supported Approved Uncertain
Intermediate filaments 1801.4141711930
Actin filaments 2401.8163614741
Focal adhesion sites 1431.111288123
Microtubules 2762.174517747
Microtubule ends 600321
Cytokinetic bridge 1601.232510923
Midbody 560.4412346
Midbody ring 300.212243
Cleavage furrow 100010
Mitotic spindle 890.73284414
Centriolar satellite 1801.4133210530
Centrosome 3873158324346
Mitochondria 11398.712038454986
Aggresome 200.200173
Cytosol 481336.932913602645479
Cytoplasmic bodies 730.6326359
Rods & Rings 190.103160
Endoplasmic reticulum 52345419225720
Golgi apparatus 11278.672199745111
Vesicles 217716.71043611427285
Peroxisomes 230.251251
Endosomes 170.17820
Lysosomes 190.151310
Lipid droplets 400.3611221
Plasma membrane 198715.21146091060204
Cell Junctions 3312.5239418529
Nucleoplasm 615547.277417763044561
Nuclear membrane 2782.1175318622
Nucleoli 10698.299239581150
Nucleoli fibrillar center 2982.3156119824
Nucleoli rim 1561.236475914
Nuclear speckles 4963.85515124743
Nuclear bodies 5864.54219630147
Kinetochore 500500
Mitotic chromosome 700.51714354
Number of proteins 130411001517463976431666

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