The multilocalizing proteome

The immunofluorescence (IF)-based approach used in the Cell Atlas allows analysis of protein distribution in all organelles and cellular substructures simultaneously. This allows for study of spatial distribution of proteins in their cellular context and identification of proteins that localize to more than one compartment, referred to as "multilocalizing proteins" (MLPs).

Figure 1 shows example images of MLPs representing common combinations of locations and gives an idea of the cellular roles of MLPs. The most common case is that MLPs are located at multiple sites at the same time, within the same cell, but there are also MLPs that are associated with cell line-specific variations. For example, ZNF554 is a solely nuclear protein in RT4 and SH-SY5Y cells, but becomes a MLP in U-2 OS due to its additional prominent location in the nucleoli.


IPO7 - A-431

RPL19 - A-431

CCDC51 - U-2 OS


KIAA1522 - HaCaT

ITM2B - RT4

ENO1 - U-2 OS

Figure 1. Examples of MLPs identified in the Cell Atlas. IPO7 mediates the import of proteins from the cytosol to the nucleus and can cross the nuclear membrane rapidly in both directions (detected in A-431 cells). RPL19 is a component of the ribosomal 60S subunit and was identified in nucleoli, where ribosomes are assembled, and in the cytosol and endoplasmic reticulum, where protein synthesis takes place (detected in A-431 cells). CCDC51 encodes an uncharacterized protein located in the mitochondria and nucleoplasm (detected in U-2 OS). KIAA1522 encodes an uncharacterized protein identified in the plasma membrane and nucleoplasm (detected in HaCaT cells). ITM2B is a transmembrane protein processed in the Golgi apparatus and vesicles. The resulting small peptide is secreted (detected in RT4 cells). ENO1 is a well described moonlighting protein. It has several functions in different compartments including a role in glycolysis in the cytosol, and as a surface protein in the plasma membrane (detected in U-2 OS cells).

MLPs in the Cell Atlas

Approximately half of the proteins localized in the Cell Atlas (55%, n=7106) are MLPs (Figure 2). Of these, around 32% (n=2269) can be found at three or more locations. The distribution of single and multilocalizing proteins for each organelle is shown in Figure 3 and Table 1. Although around half of the human proteome consists of MLPs, the percentage of MLPs in the individual organelle proteomes is often much higher, because of the double counting of MLPs. Organelles such as the plasma membrane, cytosol, nucleus, and nucleoli share the majority of their proteins with other subcellular structures. This may reflect a need for proteins that operate across the borders of these organelles in order to regulate metabolic reactions or gene expression, or to transmit information from the surrounding environment. In contrast, the proteomes of mitochondria and the endoplasmic reticulum contain mainly single localizing proteins, suggesting that these compartments are more self-contained with regards to their biological function.

Figure 2. Bar plot showing the number of protein-coding genes for single or multilocalizing proteins.

Figure 3. Bar plot showing the distribution of proteins localized to one or multiple organelles. Note that proteins localized to different substructures of organelles (e.g. nuclear bodies and nucleoplasm) are considered multilocalizing.

Table 1. Detailed information about single and multilocalizing proteins in the proteome of organelles and substructures.

Location Number of additional protein locations
0%1% 2% 3 or more%
Actin filaments 3213933988372511
Aggresome 00167641915
Centriolar satellite 2716804742252213
Centrosome 93251223213436287
Cleavage furrow 0000150150
Cytokinetic bridge 21463064424227
Cytoplasmic bodies 13174356192523
Cytosol 966212222481206262646
Focal adhesion sites 2517523655381410
Intermediate filaments 57316937502795
Microtubule ends 00233117350
Microtubules 6525913575292911
Midbody 611193419341221
Midbody ring 275181761414
Mitochondria 599523803314613313
Mitotic spindle 00111342483540
Rods & Rings 42094563015
Kinetochore 0000250250
Mitotic chromosome 00162430452131
Nuclear bodies 771327046176306010
Nuclear membrane 4115134488129218
Nuclear speckles 20141191398117173
Nucleoli 909466463353311712
Nucleoli fibrillar center 4515150508629196
Nucleoli rim 64342169435031
Nucleoplasm 1789292740451316212805
Cell Junctions 521612339111353110
Endoplasmic reticulum 23348155327816174
Endosomes 42495331816
Golgi apparatus 266244324033431605
Lipid droplets 1333215351313
Lysosomes 3151155420210
Peroxisomes 15657301400
Plasma membrane 3121779442615331619
Vesicles 6693284341458221085

The number of MLPs is large. To get a better overview of the multilocalizing proteome, organelles can be grouped into three meta-compartments, and genes encoding MLPs can be aligned on a circular plot (Figure 4). The meta-compartments are "Nucleus" (nuclear and nucleolar structures), "Cytoplasm" (cytosol, mitochondria, and the different types of cytoskeleton), and "Secretory Pathway" (endoplasmic reticulum, Golgi apparatus, vesicles, plasma membrane). This reveals subordinate organization patterns of the MLPs. For instance, for the meta-compartments cytoplasm and nucleus, a common pattern is multilocalization between the predominant organelles cytosol and nucleoplasm, respectively. There are also many proteins that localize to more than one of the fine substructures within each of these meta compartments. The MLPs in the secretory pathway exhibit a more sequential pattern likely reflecting the directional protein trafficking. In addition, the secretory pathway shares a strikingly high number of MLPs with the nucleus, despite them not being in direct physical contact with each other. In agreement, cytoscape plots of each organelle (Figure 5, at end of the page) show that dual locations to the nucleoplasm together with the Golgi apparatus or vesicles are indeed overrepresented. This suggests that the proteomes of organelles in the secretory pathway are more versatile and should not be simplified to their role in protein secretion.


Figure 4. Circular plot with the identified proteins of each compartment presented and sorted by meta-compartments (red: Nucleus, blue: Cytoplasm, yellow: Secretory Pathway). Multilocalizing proteins appearing more than once in the plot are connected by a line

Why does the cell have MLPs?

MLPs present several advantages for the cell, some of which are crucial for cell survival. Shuttling proteins constantly switch their location in order to transport other proteins between organelles, making their multilocalization inseparably tied to their function. For example, members of the importin family transport proteins from the cytosol to the nucleus and hence are found in both organelles (Lange A et al. (2007), see also Figure 1). Another advantage of multilocalization is the possibility to make use of the same proteins in similar cellular processes and reactions, even if they occur in different subcellular compartments. For example, it has been shown that mitochondria and peroxisomes share some enzymes in their lipid metabolism (Ashmarina LI et al. (1999)). A switch of the subcellular location can also be an important way of generating a quick cellular response upon environmental changes, and external or internal cues. For example, receptors such as ERBB2 located in the plasma membrane are known to move to the nucleus after stimulation, where they change the expression pattern of target genes. This translocation has a profound impact on cancer initiation, progression, and prognosis in human cancers (Wang SC et al. (2009)).

Some of the MLPs are not just multilocalizing, but also multifunctional proteins. Multifunctional proteins do not fit in the paradigm of "one gene - one protein - one function", and certainly adds another dimension to cellular complexity. Multifunctionality may be the result of eg. gene fusions, expression of several splice variants, different post-translational modifications, different interaction partners and/or multilocalization of the protein. An extreme group of multifunctional proteins are the moonlighting proteins. The term "moonlighting" has been used for people who work in different jobs during daylight and moonlight, and like their human counterpart, moonlighting proteins have two or more completely different biochemical functions (Jeffery CJ. (1999)). Moonlighting proteins may provide connections and switches between different cellular reactions, pathways and processes, making it possible for cells to coordinate responses to a changing environment (Jeffery CJ. (2015)). For example, some biosynthetic enzymes moonlight as transcription factors, in order to provide a feedback-loop for transcription of genes involved in the pathway. An example of a moonlighting and multilocalizing protein is ENO1 (Figure 1) that acts a glycolytic enzyme in the cytosol, but also as a plasminogen-receptor in the plasma membrane, and as a transcriptional repressor in the nucleus (Pancholi V. (2001)).

The Human Protein Atlas does not provide functional studies of proteins and therefore cannot determine if a MLP is multifunctional. However, the description of proteins at multiple locations is an important step in the discovery of multifunctional and moonlighting proteins and the spatial information provided by the Cell Atlas could be integrated into existing prediction models (Chapple CE et al. (2015)).

Actin Filaments
Centrosome
Cytosol
Endoplasmic Reticulum
Golgi Apparatus
Intermediate Filaments
Microtubules
Mitochondria
Nuclear membrane
Nucleoli
Nucleoplasm
Plasma Membrane
Vesicles

Figure 5. Cytoscape plots of MLPs. The interactive and clickable plots show the number of all shared MLPs between the individual organelles, including MLPs with additional locations. Only connecting nodes containing more than one protein and at least 0.5% of all human proteins are shown. The circle sizes are related to the number of proteins. The cyan colored nodes show combinations that are significantly overrepresented, while magenta colored nodes show combinations that are significantly underrepresented as compared to the probability of observing that combination based on the frequency of each annotation and a hypergeometric test (p≤0.05). Each node is clickable and results in a list of all proteins that are found in the connected organelles.

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