The multilocalizing proteome

The immunofluorescence (IF)-based approach used in the subcellular section allows the analysis of protein distribution in all organelles and cellular substructures 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 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 - U2OS

KIAA1522 - HaCaT
ITM2B - RT-4
ENO1 - U2OS

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

More than half of the proteins localized in the subcellular section (57%, n=7413) are MLPs (Figure 2). Of these, around 32% (n=2393) 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. 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 mainly single localizing proteins, suggesting that this compartment is more self-contained with regards to its 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.

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 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 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 cpoupled 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 section could be integrated into existing prediction models (Chapple CE et al. (2015)).

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 more than one protein and at least 0.5% of all human proteins 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.

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