The endoplasmic reticulum (ER) is a delicate membranous network composed of sheets and tubules that spreads throughout the whole cytoplasm and is contiguous to 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, especially for protein biosynthesis and production of lipids.
The biological function of an organelle is defined by its proteome (see Figure 1 for examples of ER-associated proteins). In the Cell Atlas, 466 (2%) of all human proteins have been experimentally shown to localize to the endoplasmic reticulum (Figure 2). Around 50% (n=235) of the ER proteins localize to other cellular compartments in addition to the ER, the most common ones being the cytosol and nucleoli. A Gene Ontology (GO)-based functional enrichment analysis of the nucleolar proteins shows enriched terms for biological processes related to protein synthesis, protein folding, protein modification, mRNA degradation and metabolic processes.
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 in 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).
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.
The structure of the endoplasmic reticulum
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 has two distinct types of structures (Figure 3): flat cisternal, often stacked sheets, and reticulated tubules that are mostly connected by three-way junctions, which result in a polygonal pattern. The different membrane-to-lumen ratios in these two structures favor a dedicated function. Sheets with their large surface are enriched by ribosomes, and hence form the so-called "rough ER", the primary location for translation. In contrast, areas in the tubules that are largely devoid of ribosomes, are called "smooth ER". The smooth ER harbors the ER exit sites, is involved in the synthesis of lipids, and interacts with other organelles via specialized contact sites (Friedman JR et al, 2011).
The function of the endoplasmic reticulum
The first and foremost function of the ER is in synthesis of proteins. About one third of all cellular proteins are translocated into the lumen or the membrane of the ER, including the majority of the secreted proteins and cell-surface proteins. The translation is initiated in the cytosol, but a signal peptide guides the nascent protein to the ER where the translation continues. Here, the newly translated proteins get in contact with a dense meshwork of ER-resident proteins. These proteins aid proper protein folding, perform post-translational modifications such as glycosylation and disulfide bond formation, and finally control the quality of the newly synthesized proteins. Proteins belonging to this group such as HSP90B1 and CANX make good markers for staining of the ER (Table 1), as they are often highly expressed (Table 2).
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 (Roussel BD et al, 2013), are linked to ER stress and an imbalance in the UPR, e.g. Parkinson's disease (Omura et al, 2013) and Alzheimer's disease (Fonseca AC et al, 2013).
Table 1. Selection of proteins suitable as markers for the endoplasmic reticulum.
Table 2. Highly expressed single localizing endoplasmic reticulum proteins across different cell lines.
The ER also contains many enzymes that are required for biosynthesis of the major lipid classes and their precursors in the cell. This includes phospholipids, cholesterol, and ceramides, which forms the backbone of all sphingolipids. Additionally, 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 mainly to the ER reflects several functions associated with this organelle. The most highly enriched terms for the GO domain Biological Process are related to protein translation, such as selenocysteine metabolic processes, and mRNA degradation as well as biosynthesis of lipids (Figure 5a). For the GO domain Molecular Function, ubiquitin-specific protease is the top term, which points to the ER function of protein degradation (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 50% (n=235) 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, the Golgi apparatus or the cytosol. The ER, the Golgi apparatus and vesicles are closely connected in the secretory pathway. Hence, proteins that are synthesized in ER, are transported through the Golgi apparatus in vesicles to other organelles or the extracellular matrix. The ER is embedded in the cytosol and proteins of the cytosol can use the surface of the ER membrane for certain function, e.g. translation by ribosomal proteins, which could explain these dual localizations. 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 or in many tissues, 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.
Relevant links and publications
Thul PJ et al, 2017. A subcellular map of the human proteome. Science.