- Protein synthesis
- Glucose 6-P
Endoplasmic reticulum is a complex membrane-bound compartment arranged in tubules and flattened cisterns interconnected and sharing the same lumen (inner space) and membrane. Endoplasmic reticulum membrane is also continuous with the outer membrane of the nuclear envelope. Tubules and cisterns are distributed trough the cytoplasm from the nuclear envelope to close to the plasma membrane, so that they can account for half of the total cellular membranes. Endoplasmic reticulum membrane is thinner (around 5 nm in thickness) than other organelle membranes because they have lipids with shorter fatty acid chains.
There two main domains in the endoplasmic reticulum, easily seeing at electron microscopy: rough and smooth endoplasmic reticulum. Rough endoplasmic reticulum forms cisternae and rather straight tubules, both covered by ribosomes (that is why the name rough), whereas smooth endoplasmic reticulum is organized in irregular tubules, with no associated ribosomes. It is striking that flattened cisternae and tubules show a similar height of around 30 to 50 nm. Furthermore, cisternae can be transformed in tubules, and the other way around, by the action of reticulon and dDP1/Yop1 proteins.
The outer membrane of the nuclear envelope can be regarded as endoplasmic reticulum, since it is physically continuous with the membrane of the endoplasmic cisternae and contains associated ribosomes which are translating mRNA. Rough and smooth endoplasmic reticulum do not usually share the same cytosolic space. This non overlapping distibrution is observed in hepatocytes, neuronas and cells synthesizing steroid hormone. However, in some regions there is not a clear segregation between both domains, and tubules with associated ribosomes are intermingled with naked tubules. The spatial distribution of the endoplasmic reticulum is set by the cytoskeleton, microtubules in animal cells and actin filaments in plant cells. However, cytoskeleton does not appear to be involved in determine the shape of the endoplasmic reticulum, neither cisternae nor tubules.
Rough endoplasmic reticulum
Rough endoplasmic reticulum is organized in tubules, more or less straight, and flattened cisternae. Sometimes, cisternae are tidily piled. The name rough is due to the electron microscopy images where ribosomes, black particles, are observed covering the endoplasmic reticulum membrane. The density of associated ribosomes influences the organization, so that a high density causes a cistern-like morphology, whereas low density are found in tubules. Cisternae and tubules coexist in the same cell, but those cell with a intense secretory activity show dense piles of cisternae, which means a highly developed rough endoplasmic reticulum.
The main function of the rough endoplasmic reticulum is the synthesis of proteins to be delivered to different places, such as the extracellular space, the lumen and membranes of several organelles that are part of the vesicular traffic, such as lysosomes, Golgi complex and endosomes, and there are also proteins which will be part of the plasma membrane. Furthermore, rough endoplasmic reticulum needs to produce proteins for itself, which are known as resident proteins. Keep in mind that a protein should "know" what is its final place, and this can be done by a signal peptide (short sequence of amino acids) or particular modifications of the protein, which are like addresses so that when a protein arrives to a station of dispatch, mainly in the trans Golgi network (TGN) of the Golgi complex, these addresses are recognized, the molecule is packaged in a particular type of vesicle and shipped to the target compartment.
Any protein to be secreted or to be part of a compartment of the vesicular traffic is synthesized in the rough endoplasmic reticulum, either remaining free in the lumen or as a part of its membrane. The process starts when a mRNA, which carries information for a protein that will be part of the vesicular traffic, joins to the large ribosomal subunit, and then to the small ribosomal subunit, to begin the translation. The first translated part of the mRNA is a sequence of amino acids known as signal peptide, which is about 70 amino acids long. This sequence is recognized by a cytosolic molecule referred as SRP (sequence recognizing peptide). SRP is a mix of 1 RNA and 6 polypeptides that joins to the signal peptide and slows very much the translation process. The mRNA-ribosome-SRP-signal-peptide complex diffuses through the cytosol until it hits a rough endoplasmic reticulum membrane. In these membranes, there are SRP-receptors which recognise SRP. The whole complex becomes attached to the membrane and then interacts with a translocon, a large transmembrane protein with a channel. Then SRP and SRP-receptor are released and mRNA-ribosome-nascent-peptide attached to the translocon can continue the translation, but the nascent polypeptide grows now inside of the channel of the translocon. The signal peptide gets fixed to the channel walls, whereas the rest of the protein is falling into the lumen of the endoplasmic reticulum cisterna. The signal peptide is removed from the rest of the polypeptide by a peptidase, and once completely translated, the new protein is released and remains free in the lumen, but quickly will adopt a spatial proper conformation helped by chaperones, another proteins. When translation is finished, the ribosome-mRNA is disengaged from the translocon, and all of them are free for another round.
If the protein to be synthesized is a membrane protein, it will not be released into the lumen of the rough endoplasmic reticulum after the translation by ribosomes. Membrane proteins contain sequences of hydrophobic amino acids, and when these hydrophobic amino acid sequences are being translated and going through the channel of the translocon, they can cross the wall of the translocon and then be among the fatty acid chains of the membrane lipids. The process is rather complex and diverse depending of the protein. For example, some receptors are transmembrane proteins with a chain of amino acids that can cross seven times the cell membrane, by means of alternating hydrophobic and hydrophilic animo acid sequences. Furthermore, there are proteins spanning just one monolayer of the membrane, and they have to be either in the cytosolic monolayer or in the monolayer facing the lumen. It is very rare, but rough endoplasmic reticulum may import some proteins synthesized in the cytosol.
Rough reticulum endoplasmic proteins are modified at the same time they are being synthesized. a) There is a glycosylation (N-glycosylation) of asparagine amino acids, which receive a carbohydrate molecular complex composed of 15 monosaccharides. This molecular complex is first assembled into dolichol phosphate, a membrane lipid, and then transferred to an asparagine of the nascent protein. Some of the terminal monosaccharides of this complex will be lost in the Golgi apparatus. b) Some proteins, particularly those targeted to the extracellular matrix, will be hydroxylated. This process happens to proline and lysine amino acids of the collagen molecules, which end up being hydroxyproline and hydroxylysine amino acids. c) Some integral proteins of the plasma membrane are chemically bonded to some membrane lipids. This chemical bond is also established in the rough endoplasmic reticulum.
A quality control of the synthesized proteins occurs in the rough endoplasmic reticulum, so that those defective proteins are removed from the reticulum and degraded in the cytosol. Proteins known as chaperones, which are needed for a proper folding of proteins, also play an essential role in detecting defective proteins. Other proteins bearing lectin domains are able to detect and recognize wrong arrangements of carbohydrates in the newly synthesized proteins. Wrong folding of nascent proteins, which may lead to cell damages, is more frequent than one may imagine. For example, around 80 % of the new CFTR (Cystic fibrosis transmembrane conductance regulator) proteins are misfolded.
As we already mentioned, proteins synthesized in the rough endoplasmic reticulum may end in different compartments: extracellular space by secretion, in the plasma membrane, or in one of the vesicular traffic organelles such as Golgi complex, endosomes and lysosomes. However, some of the proteins remain in the endoplasmic reticulum. These are known as resident proteins. We already learnt some of them: chaperones, SRP receptor, translocon, some glucosidases, and there are many others. To be retained in the endoplasmic reticulum, these proteins should have a four amino acid sequence located in the carboxyl end (-COOH) of the protein.
Smooth endoplasmic reticulum
Smooth endoplasmic reticulum is a network of interconnected tubules, which are continuous with the rough endoplasmic reticulum. There are no ribosomes associated to its membrane, that is why the name of smooth. Because of that, all proteins needed to work in this compartment come from the rough endoplasmic reticulum. Smooth endoplasmic reticulum is abundant in those cells involved in lipid metabolism or detoxification, and is also an organelle for calcium storage.
Salient functions of the smooth endoplasmic reticulum are:
Most of the membrane lipids are synthesized in the smooth endoplasmic reticulum, including glycerophospholipids and cholesterol. Most of the sphingolipid synthesis, however, happens in the Golgi complex, but the ceramide, the basic component of sphingolipids, is synthesised in the smooth endoplasmic reticulum. Not all of the synthesis process of glycerophospholipids happens in the smooth endoplasmic reticulum membranes, since the fatty acids come from the cytosol and are inserted in the membrane of the organelle, where they are then transformed in glycerophospholipids. Lipid synthesis is carried out by membrane proteins with the active site facing the cytosol, hence lipids are located initially in the cytosolic monolayer of the smooth endoplasmic reticulum membrane. Because flip-flop movement is mostly forbiden for lipids by the hydrophobic environment of fatty acid chains, lipids need a help to go to the other monolayer (facing the lumen). There are specialized proteins that can change the lipids from one monolayer to the other: flipases, flopases and scramblases. Flipases move lipids from de cytosolic monolayer to the lumen monolayer (or the extracellular space in the plasma membrane), whereas flopasases move lipids in the other way around. Scramblases, however, can move lipids in both directions. These proteins can be found in almost every membrane of the cell. In the endoplasmic reticulum, both monolayers show similar lipid composition, so that the asymmetry found in other membranes of the cell are due to the activity of these interchange proteins, which depending on the membrane may be of different type and number. However, the carbohydrates of the plasma membrane, which contribute to the membrane asymmetry, start to be synthesized in the endoplasmic reticulum, and are later modified in the Golgi complex.
Cholesterol, which is synthesized in the smooth endoplasmic reticulum, is another important molecule for membranes, particularly for the plasma membrane. Cholesterol is transported by vesicles or molecular from the endoplasmic reticulum to the plasma membrane. A diverse set of transporters is the main way to move ergosterol from one membrane to another in yeasts (they have ergosterol instead of cholesterol). This transport does not need ATP.
Mitochondria, chloroplast and peroxisomes are not part of the vesicular traffic, so their membrane lipids should be imported or synthesized locally. Molecular carriers are used for this transport. For example, glycerophospholipids are moved by a cytosolic proteins known as glycerophospholipid interchangers, which can move this type of lipids through the cytosol. They can take the lipids from the membrane of the smooth endoplasmic reticulum and drop them in the membrane of other organelle. Furthermore, many electron microscopy images show physical contacts between membranes of different organelles, for example between endoplasmic reticulum and mitochondria or peroxisomes. This contacts may facilitate direct interchange of lipids between different membranes. Chloroplasts can synthesize their own glycerophospholipids and glycolipids.
Triacylglycerols are also synthesized in the smooth endoplasmic reticulum. These lipids are stored in the reticulum itself or as lipid drops. This synthesis is very active in the adipocytes, fat storage cells, for two purposes: energy storage and thermal insulation. Triacylglycerols are also part of the lipoproteins, and are needed for the production of steroid hormones and biliary acids.
Hepatocytes, liver cells, show a highly developed smooth endoplasmic reticulum. They synthesize lipoproteins for transporting cholesterol and other lipids to all parts of the body. Furthermore, in the smooth endoplasmic reticulum membranes, the P450 protein family is in charge of removing potentially toxic metabolites, as well as liposoluble toxins incorporated during digestion. The smooth endoplasmic membranes enlarge to make room to all these enzymes, which in turn depends on how many toxics the animal body contain.
Dephosphorylation of 6-phosphate glucose.
Glucose is usually stored as glycogen, mainly in the liver. This organ delivers glucose to blood, process regulated by two hormones: insulin and glucagon. Catabolism of glycogen produces 6-phosphate glucose, which can not cross the cell membrane and hence can not leave the cell. Glucose 6-phosphatase, which is anchored to the endoplasmic reticulum, removes the phosphate residue allowing glucose to be moved out of the cell.
Smooth endoplasmic reticulum also works as a storage space for cytosolic calcium. Stored calcium is released as a consequence of extracellular or intracellular signals acting via second messengers. A remarkable example is the endoplasmic reticulum of the muscle cells (known as sarcoplasmic reticulum), which is able to capture cytosolic calcium by a calcium pump located in its membrane. Capture and release of calcium happen in every muscle cell contraction cycle.
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