The multilocalizing proteome

The immunofluorescence (IF)-based approach used in the subcellular resource enables analysis of protein distribution in all organelles and subcellular structures simultaneously. This allows for the 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 nuclear protein in RT4 and SH-SY5Y cells, but becomes a MLP in U-2 OS due to it's additional prominent location in the nucleoli.


IPO7 - A-431

RPL19 - A-431

CCDC51 - U2OS


KIAA1522 - HaCaT

ITM2B - RT-4

ENO1 - U2OS

Figure 1. Examples of MLPs identified in the subcellular resource. 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 subcellular resource

More than half of the proteins localized in the subcellular resource (60%, n=8187) are MLPs (Figure 2). Of these, around 38% (n=3112) can be found in three or more locations. The distribution of single and multilocalizing proteins for each major organelle is shown in Figure 3 and Table 1. The percentage of MLPs in the individual organelle proteomes varies, but is often more than half 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, control gene expression, and/or to transmit information from the surrounding environment. In contrast, the proteomes of mitochondria contain a larger fraction of single localizing proteins, suggesting that this compartment is more self-contained with regards to its biological function.

0.01.0k2.0k3.0k4.0k5.0k6.0k7.0k8.0k9.0k10k11k12k13kNumber of genes4 or more organelles3 organelles2 organelles1 organelle

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

Nuclear MembraneNucleoplasmNucleoliCytosolPlasma MembraneMitochondriaEndoplasmic ReticulumGolgi ApparatusVesiclesMicrotubulesCentrosomeActin FilamentsIntermediate FilamentsPrimary CiliumSperm0.05001.0k1.5k2.0k2.5k3.0k3.5k4.0k4.5kSingle localizingMultilocalizing

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 2410823384335622
Aggresome 001257629314
Centriolar satellite 2410652850226629
Centrosome 5211109231523213628
Cleavage furrow 0000002100
Cytokinetic bridge 21361748228640
Cytoplasmic bodies 15203547182457
Cytosol 9001722424313232660012
Focal adhesion sites 2618432946322819
Intermediate filaments 44296342352375
Microtubule ends 0000343457
Microtubules 37117923641910731
Midbody 59173212231732
Midbody ring 283121248624
Mitochondria 536483723315013535
Mitotic spindle 0011830225843
Rods & Rings 315115552515
Cell Junctions 471412438103314413
Endoplasmic reticulum 23141201369817366
Endosomes 319956213213
Golgi apparatus 27021491393462812210
Lipid droplets 1128215351338
Lysosomes 151158421316
Peroxisomes 15637291414
Plasma membrane 33415853396583031114
Vesicles 68028922385262223410
Kinetochore 00114229343
Mitotic chromosome 00172333442432
Nuclear bodies 661125341196329015
Nuclear membrane 32111194190314516
Nuclear speckles 17836183378718469
Nucleoli 10610457413613316315
Nucleoli fibrillar center 40131454593293812
Nucleoli rim 75291963425033
Nucleoplasm 1603262636421409235459
Basal body 20307741822255
Primary cilium 003810621620553
Primary cilium tip 0035123767
Primary cilium transition zone 008914165563
Acrosome 118231734264332
Annulus 125109182550
Calyx 345711163246
Connecting piece 005514154852
End piece 1110525138142
Equatorial segment 00141714173948
Flagellar centriole 222218165750
Mid piece 185298571616748
Perinuclear theca 1134234059
Principal piece 1443810812114939

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 the nucleus (nuclear and nucleolar structures shown in red), the cytoplasm (cytosol, mitochondria, and the different types of cytoskeleton shown in blue), and the secretory pathway (endoplasmic reticulum, Golgi apparatus, vesicles, plasma membrane shown in yellow). 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 that they are not in direct physical contact with each other. In agreement, cytoscape plots of each organelle (Figure 5, at the 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 for 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, or other 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 of 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 tightly coupled feedback loop for transcription of genes involved in the pathway. An example of a moonlighting and multilocalizing protein is ENO1 (Figure 1) that acts as 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 in the subcellular resource 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
Primary Cilium
Sperm
Vesicles

Figure 5. Cytoscape plots showing the distribution of MLPs that are shared between the major organelle proteomes. The black middle node links to all proteins localizing to the selected major organelle proteome, while the gray nodes links to all MLPs shared with each of the other major organelle proteomes. Only gray nodes with at least 1 % of the proteins in the compartment are shown. The colored connecting nodes show the number of proteins that are exclusively shared between the compartments. The circle sizes of the connecting nodes are related to the number of proteins exclusively shared between the compartments. 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 link to a list of the corresponding genes.

Relevant links and publications

Lange A et al., Classical nuclear localization signals: definition, function, and interaction with importin alpha. J Biol Chem. (2007)
PubMed: 17170104 DOI: 10.1074/jbc.R600026200

Ashmarina LI et al., 3-Hydroxy-3-methylglutaryl coenzyme A lyase: targeting and processing in peroxisomes and mitochondria. J Lipid Res. (1999)
PubMed: 9869651 

Wang SC et al., Nuclear translocation of the epidermal growth factor receptor family membrane tyrosine kinase receptors. Clin Cancer Res. (2009)
PubMed: 19861462 DOI: 10.1158/1078-0432.CCR-08-2813

Jeffery CJ., Moonlighting proteins. Trends Biochem Sci. (1999)
PubMed: 10087914 

Jeffery CJ., Why study moonlighting proteins? Front Genet. (2015)
PubMed: 26150826 DOI: 10.3389/fgene.2015.00211

Pancholi V., Multifunctional alpha-enolase: its role in diseases. Cell Mol Life Sci. (2001)
PubMed: 11497239 DOI: 10.1007/pl00000910

Chapple CE et al., Extreme multifunctional proteins identified from a human protein interaction network. Nat Commun. (2015)
PubMed: 26054620 DOI: 10.1038/ncomms8412