The endoplasmic reticulum (ER) is a delicate membranous network composed of sheets and tubules that spread throughout the cytoplasm and are contiguous with the nuclear membrane. The expanded surface of the ER membrane as well as the distinct composition of the ER lumen provides a platform for various biochemical reactions, but especially for protein biosynthesis and production of lipids.
In the Cell Atlas, 483 genes (2% of all protein-coding human genes) have been shown to encode proteins that localize to the ER (Figure 2). Around 52% (n=250) of the ER proteins localize to other cellular compartments in addition to the ER, the most common ones being the cytosol and vesicles. A Gene Ontology (GO)-based functional enrichment analysis of the ER proteins shows enriched terms for biological processes related to protein synthesis, protein folding, protein modification, mRNA degradation and metabolic processes. Examples of ER-associated proteins can be found in Figure 1.
Figure 1. Examples of proteins localized to the endoplasmic reticulum. ELOVL5 is an ER membrane protein that catalyzes the first and rate limiting reaction in the elongation of long and very long-chain polyunsaturated fatty acids (detected in A-431 cells). STIM1 is a transmembrane protein that is involved in the regulation of calcium ions (detected in A549 cells). VAPA may regulate the morphology of the ER by interacting with the cytoskeleton (detected in A-431 cells).
2% (483 proteins) of all human proteins have been experimentally detected in the endoplasmic reticulum by the Human Protein Atlas.
236 proteins in the endoplasmic reticulum are supported by experimental evidence and out of these 53 proteins are enhanced by the Human Protein Atlas.
250 proteins in the endoplasmic reticulum have multiple locations.
66 proteins in the endoplasmic reticulum show a cell to cell variation. Of these 64 show a variation in intensity and 2 a spatial variation.
Proteins localizing to the ER are mainly involved in protein synthesis, protein folding, protein modification, mRNA degradation and metabolic processes.
Figure 2. 2% of all human protein-coding genes encode proteins localized to the endoplasmic reticulum. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Figure 3. Examples of the morphology of the ER in different cell lines, represented by immunofluorescent staining of the protein encoded by LRRC59 in U-2 OS, U-251 MG, and A-431 cells.
The ER is a large and dynamic structure (Schwarz DS et al. (2016), made up of flat cisternal, often stacked sheets, and reticulated tubules, which are mostly connected by three-way junctions, which result in a polygonal pattern. The different membrane-to-lumen ratios in these two domains reflect their different functions. The sheets with their large surface are studded with ribosomes, forming the "rough ER", and is the primary location for translation. In contrast, areas in the tubules are largely devoid of ribosomes, forming the "smooth ER". and is involved in many other functions, depending on cell type. (Friedman JR et al. (2011)). There are also areas that are partially smooth and partially rough, referred to as the transitional ER.
Proteins that are suitable as markers for the ER can be found in Table 1. Highly expressed genes encoding proteins that localize to the ER are listed in Table 2.
Table 1. Selection of proteins suitable as markers for the endoplasmic reticulum.
Figure 4. 3D-view of the ER in U-2 OS,visualized by immunofluorescent staining of HSP90B1. The morphology of the ER in human induced stem cells can be seen in the Allen Cell Explorer.
The function of the endoplasmic reticulum
The ER is known to serve multiple roles in human cells (Schwarz DS et al. (2016). One of the major functions of ER is in translation of mRNA to certain groups of proteins, including secreted proteins and integral membrane proteins, but also some cytosolic proteins. Translation of these proteins is initiated in the cytosol, but the emergence of an N-terminal signal peptide guides the nascent protein and the ribosome to SRP receptors in the rough ER. After docking to the ER, translation continues and the nascent protein gets translocated across the ER membrane through channels referred to as translocons. If a transmembrane domain is present, the protein gets incorporated in the lipid bilayer of the ER. The ER lumen contains proteins that mediate proper protein folding, post-translational modifications and quality control of the newly synthesized proteins. Proteins that are targeted for other parts of the secretory pathway, the plasma membrane or other organelles begin the process of transport from the ER, using exit sites present in the transitional ER.
Only correctly folded proteins are transported out of the ER. Unfolded or misfolded proteins can cause ER stress by accumulating in the lumen. This process activates the unfolded protein response (UPR), which resolves the stress by reducing the overall protein synthesis, increasing the capacity for protein folding, and promoting the removal of misfolded proteins by the ER-associated degradation (ERAD) (Travers KJ et al. (2000)). However, if the stress is not alleviated, it ultimately induces apoptosis. Several pathological processes, especially neurological diseases like Parkinson's- and Alzheimer's disease, have been linked to ER stress and an imbalance in the UPR (Roussel BD et al. (2013)).
The smooth ER contains enzymes involved in carbohydrate metabolism, gluconeogenesis, and lipid biosynthesis. The latter include synthesis of the phospholipids, which are the major lipid components of cellular membranes. In addition, the smooth ER harbours most of the cytochrome P450 enzymes that are involved in metabolism of a variety of endogenous and exogenous toxic compounds (Neve EP et al. (2010)). Moreover, the ER lumen is one of the major storage sites of intracellular calcium ions and maintains the Ca2+ homeostasis by a controlled release and uptake of the ions.
Gene Ontology (GO)-based enrichment analysis of genes encoding proteins that localize to the ER highlight several functions associated with this organelle. The most highly enriched terms for the GO domain Biological Process are related to protein translation, mRNA degradation, biosynthesis of lipids and metabolic processes (Figure 5a). For the GO domain Molecular Function, there is an enrichment of ubiquitin-specific protease activity, which points to the function of the ER in protein degradation, RNA binding, transferase activity and certain enzymatic activities (Figure 5b).
Figure 5a. Gene Ontology-based enrichment analysis for the endoplasmic reticulum proteome showing the significantly enriched terms for the GO domain Biological Process. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Figure 5b. Gene Ontology-based enrichment analysis for the endoplasmic reticulum proteome showing the significantly enriched terms for the GO domain Molecular Function. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Endoplasmic reticulum-associated proteins with multiple locations
In the Cell Atlas, approximately 52% (n=250) of the annotated ER proteins also localize to other compartments in the cell. The network plot (Figure 6) shows an overrepresentation for proteins localized to the ER together with vesicles and/or the cytosol. This likely reflects the close connection of the ER and vesicles in the secretory pathway. Similarly, proteins that localize to the cytosol, can interact with the ER as part of certain functions, the most prominent example being the recruitment of ribosomal proteins to the ER during translation of certain groups of proteins. Examples of multilocalizing proteins within the ER proteome can be seen in Figure 7.
Figure 6. Interactive network plot of ER proteins with multiple localizations. The numbers in the connecting nodes show the proteins that are localized to the ER and to one or more additional locations. Only connecting nodes containing more than one protein and at least 0.5% of proteins in the ER proteome 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). Note that this calculation is only done for proteins with dual localizations. Each node is clickable and results in a list of all proteins that are found in the connected organelles.
Figure 7. Examples for multilocalizing proteins in the endoplasmic reticulum proteome. CREB3L2 is an ER membrane protein, whose cytosolic N-terminal domain is translocated to the nucleus upon ER-stress (detected in U-2 OS cells). LPCAT2 was found in both the ER and lipid droplets. The ER has a direct role in the emergence and regression of lipid droplets and many RPL28 encodes a component of ribosomes and is required for protein biosynthesis in both ER and cytosol (detected in U-2 OS cells).
Expression levels of endoplasmic reticulum proteins in tissue
Transcriptome analysis and classification of genes into tissue distribution categories (Figure 8) shows that genes encoding ER-associated proteins are more likely to be detected in all tissues, and less likely to be detected in a single tissue, compared to all genes presented in the Cell Atlas. This indicates that a large fraction of the ER-associated proteins are likely to fulfill housekeeping functions needed in all tissue types.
Figure 8. Bar plot showing the percentage of genes in different tissue distribution categories for endoplasmic reticulum-associated protein-coding genes, compared to all genes in the Cell Atlas. Asterisk marks a statistically significant deviation (p≤0.05) in the number of genes in a category based on a binomial statistical test. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Menon M et al., Single-cell transcriptomic atlas of the human retina identifies cell types associated with age-related macular degeneration.Nat Commun. (2019)
PubMed: 31653841 DOI: 10.1038/s41467-019-12780-8
Wang L et al., Single-cell reconstruction of the adult human heart during heart failure and recovery reveals the cellular landscape underlying cardiac function.Nat Cell Biol. (2020)
PubMed: 31915373 DOI: 10.1038/s41556-019-0446-7
Wang Y et al., Single-cell transcriptome analysis reveals differential nutrient absorption functions in human intestine.J Exp Med. (2020)
PubMed: 31753849 DOI: 10.1084/jem.20191130
Stadler C et al., Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy.J Proteomics. (2012)
PubMed: 22361696 DOI: 10.1016/j.jprot.2012.01.030
Poser I et al., BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.Nat Methods. (2008)
PubMed: 18391959 DOI: 10.1038/nmeth.1199
Bird RP., Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings.Cancer Lett. (1987)
PubMed: 3677050 DOI: 10.1016/0304-3835(87)90157-1
HUXLEY AF et al., Structural changes in muscle during contraction; interference microscopy of living muscle fibres.Nature. (1954)
HUXLEY H et al., Changes in the cross-striations of muscle during contraction and stretch and their structural interpretation.Nature. (1954)
Cho RJ et al., Transcriptional regulation and function during the human cell cycle.Nat Genet. (2001)
PubMed: 11137997 DOI: 10.1038/83751
Whitfield ML et al., Identification of genes periodically expressed in the human cell cycle and their expression in tumors.Mol Biol Cell. (2002)
PubMed: 12058064 DOI: 10.1091/mbc.02-02-0030.
Boström J et al., Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells.PLoS One. (2017)
PubMed: 29228002 DOI: 10.1371/journal.pone.0188772
Lane KR et al., Cell cycle-regulated protein abundance changes in synchronously proliferating HeLa cells include regulation of pre-mRNA splicing proteins.PLoS One. (2013)
PubMed: 23520512 DOI: 10.1371/journal.pone.0058456
Ohta S et al., The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics.Cell. (2010)
PubMed: 20813266 DOI: 10.1016/j.cell.2010.07.047
Ly T et al., A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells.Elife. (2014)
PubMed: 24596151 DOI: 10.7554/eLife.01630
Pagliuca FW et al., Quantitative proteomics reveals the basis for the biochemical specificity of the cell-cycle machinery.Mol Cell. (2011)
PubMed: 21816347 DOI: 10.1016/j.molcel.2011.05.031
Ly T et al., Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.Elife. (2015)
PubMed: 25555159 DOI: 10.7554/eLife.04534
Dueck H et al., Variation is function: Are single cell differences functionally important?: Testing the hypothesis that single cell variation is required for aggregate function.Bioessays. (2016)
PubMed: 26625861 DOI: 10.1002/bies.201500124
Snijder B et al., Origins of regulated cell-to-cell variability.Nat Rev Mol Cell Biol. (2011)
PubMed: 21224886 DOI: 10.1038/nrm3044
Cooper S et al., Membrane-elution analysis of content of cyclins A, B1, and E during the unperturbed mammalian cell cycle.Cell Div. (2007)
PubMed: 17892542 DOI: 10.1186/1747-1028-2-28
Davis PK et al., Biological methods for cell-cycle synchronization of mammalian cells.Biotechniques. (2001)
PubMed: 11414226 DOI: 10.2144/01306rv01
Scialdone A et al., Computational assignment of cell-cycle stage from single-cell transcriptome data.Methods. (2015)
PubMed: 26142758 DOI: 10.1016/j.ymeth.2015.06.021
Sakaue-Sawano A et al., Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.Cell. (2008)
PubMed: 18267078 DOI: 10.1016/j.cell.2007.12.033
Grant GD et al., Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors.Mol Biol Cell. (2013)
PubMed: 24109597 DOI: 10.1091/mbc.E13-05-0264
Semple JW et al., An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes.EMBO J. (2006)
PubMed: 17053779 DOI: 10.1038/sj.emboj.7601391
Kilfoil ML et al., Stochastic variation: from single cells to superorganisms.HFSP J. (2009)
PubMed: 20514130 DOI: 10.2976/1.3223356