What are synthesized proteins
Mail traffic in the cell
Transport processes are very important in our daily life. We send letters and parcels, hand in our suitcases at the airport counter for automatic transport to the destination. The suitcase will be given an address, and if we're lucky, it will arrive at the desired location. Extensive logistics behind the scenes are necessary to forward a piece of luggage from station to station. There are similar transport processes in every cell in our body. Proteins have to be sent from their place of synthesis, the cytosol, to different places within the cell or out of the cell to different places in the body. Bernhard Dobberstein, now at the Center for Molecular Biology at Heidelberg University, decoded parts of the cell's delivery system.
Proteins have to overcome major obstacles in their journey out of the cell: They have to penetrate the cell membrane, which normally protects the cell from intruders. The cells of the body also have to cross membranes, which form numerous compartments in which different biochemical processes take place, such as energy generation or waste recycling, "export control and packaging. How do the body cells cope with these difficulties? How do they sort out the proteins that are used for The research of many working groups, including mine, has shown in the last 20 years that the cells use a similar transport logistics as the post office: The goods to be transported are given an "address" that is deciphered at various stations, The goods are then "decoded" according to their address. How the cells mark their proteins for export and then transport them through a membrane, and how we succeeded in deciphering the "delivery system" in the cell , I would like to describe in the following.
It was known many years ago that every cell in our body has the ability to export certain proteins. These can be messenger substances, hormones that transmit information between cells, as well as antibodies that circulate in the blood and protect us against intruders, or digestive enzymes that break down our food in the intestines. The latter are synthesized in cells of the pancreas, packed in transport containers, vesicles, and discharged from the cell if necessary. Since the cells of the pancreas are specialized in the secretion of proteins, it was natural to examine the "dispatch" of proteins in these cells more closely. The development of the electron microscope and the ultracentrifuge were of pioneering importance in the study of protein secretion. With the electron microscope, George Palade and his colleagues succeeded for the first time in making membrane structures inside cells visible. In the secretory cells of the pancreas they found particularly extensive membrane systems, membranes lying flat on top of one another with a rough surface, "the endoplasmic reticulum", ER, in the basal part, and above a collection of electron-dense, round structures. Since the secretory cells of the pancreas are "specialized export factories", the investigators assumed that the structures visible in the electron microscope have something to do with protein transport. In order to follow the path of secretion proteins within a pancreatic cell, they offered the cells radioactively labeled amino acids as food At various time intervals, Palades employees determined the location of the radioactively labeled proteins using “autoradiography.” This involves coating thin sections of cells with a radiation-sensitive emulsion in which the radioactive decay caused by the labeled amino acids causes blackening As with the exposure of a film, the path through the cell can be followed: five to ten minutes after the "feeding" the newly synthesized radioactively labeled proteins could be found in the rough endoplasmic reticulum, after about ten to 30 minutes ten in the flat membrane compartments in the middle part of the cell, the so-called "Golgi complex", and finally in the electron-dense round membrane structures, the "secretion granules" in the upper part of the cell. After about an hour, the proteins could be detected outside the cell. A biochemical approach confirmed the intracellular transport route of secretion proteins. Here, tissue of the pancreas was comminuted in a homogenizer, a kind of mixer, and the various membrane spaces, the organelles, were separated from one another by means of ultracentrifugation. With this method, too, newly synthesized secretion proteins were found first within the membrane spaces of the endoplasmic reticulum and later in the round secretion granules. Palade received the Nobel Prize in Medicine in 1974 for his work on the secretory pathway of proteins.
I want to trace the delivery system of the cell one more time. The path begins with the synthesis of a protein on the ribosomes, the "protein factories" of the cell. When a ribosome synthesizes a secretory protein, it sits on the membrane of the endoplasmic reticulum, hence the impression "Drauh" in the electron microscope. The ribosomes bind to the membrane on the one hand via a direct interaction and on the other hand via the newly synthesized protein strand that was already being synthesized during its synthesis In the interior of the ER, its "lumen", the newly synthesized protein strand is received by a large number of helpers, the "chaperones." They play an essential role in the complicated folding of the linear protein strand into its final form three-dimensional structure. Correct folding is essential for systematic functioning and is subject to strict quality control. Only proteins that pass the control are packaged in transport containers, membrane vesicles, and brought to the next station of the secretory pathway, the Golgi complex System of flat, one on top of the other many newly synthesized secretion proteins are modified by rearrangement and attachment of sugars and then collected and stored in secretion containers. When food arrives in the stomach, messenger substances rush to the pancreas, which cause the secretion containers to fuse with the plasma membrane and the stored secretion proteins are released.
The main features of the secretory path were known from the work of Palade and his colleagues at the end of the 1970s. The question of how the cell determines which proteins are intended for export and which should remain in the cell remained open. Many hypotheses have been put forward, including the signal hypothesis formulated in 1971 by Günter Blobel, Rockefeller University, New York, which states that secretory proteins themselves must have a signal sequence at their beginning, a kind of zip code, which is from a receptor in the membrane of the endoplasmic reticulum would be recognized. Only if a protein were characterized as a real secretion protein would the path through the membrane be free. Proteins that remain in the cell should not have a signal sequence and proteins that are transported to other cell organelles should have a signal sequence with different properties than those of the secretory proteins. At the time, I was working as a postdoc in Blobel's laboratory and was enthusiastic about the signal hypothesis. It was exciting to imagine that a cell exports proteins according to a sorting principle similar to how the post office sends letters. But how could we prove experimentally whether this hypothesis was right or wrong? We decided to trace the transport of a protein through the membrane of the endoplasmic reticulum on isolated membranes in the reagent vessel. We prepared the rough membranes of the endoplasmic reticulum from the pancreas of an experimental animal. When the tissue is homogenized, the flat membrane layers break, and thanks to the lipids in the membrane pieces, closed, round containers, called "microsomes", are formed, which carry the ribosomes on the outer surface. We used this to test whether there was also a foreign secretion protein, for example an antibody molecule that can be transported across the microsomal membrane and how globin would behave, an intracellular, cytosolic protein of red blood cells. To do this, we synthesized the radioactively labeled antibody chain and globin in a test tube in the presence and absence of microsomes. To find out whether Since the secretory protein has actually crossed the membrane and got into the membrane vesicle, we added a mixture of protein-degrading enzymes, proteases, to the approach.We knew that freely accessible proteins that were not in closed organelles would be almost completely degraded as a result which tran in the microsomes Protected proteins exercised by the surrounding membrane should remain intact. The result of this experiment was clear: only the secretory protein, the antibody chain, was protected against the degrading enzymes, i.e. it was transported through the membrane into the microsomes. Globin, on the other hand, the cytosolic protein, could be broken down by the proteases because it apparently did not get into the microsomes. However, transport only took place if the membranes were present during the synthesis of the protein, but not if the finished protein was only later brought together with microsomes. During transport through the membrane, a short section of the amino acid chain of the secretory protein was cleaved off. Later it turned out that this section contains the signal sequence, so to speak the postcode. Signal sequences have since been identified for a variety of secretory proteins. Their postcodes consist of short sections, about 15 to 40 amino acids long, all of which have a central region of hydrophobic, i.e. water-repellent, amino acids. After such signal sequences were initially found in animal cells, they could also be detected in bacterial secretion proteins. Bacteria as well choose the proteins for export by means of a signal sequence. Many signals are even interchangeable between bacterial and animal proteins. If such a signal sequence is coupled to a protein that normally remains in the cytoplasm, even this protein can be released from the cell. Here A comparison to the postal delivery system comes to mind, which is based on postal codes for the delivery of letters and parcels and transports everything if it is correctly addressed and we pay for the transport. By the way, the cell "pays" too, it has to use energy as we'll see later, just during its production, a protein is transported across the membrane of the endoplasmic reticulum. The question arises as to how the complex of ribosome and growing polypeptide chain attaches itself to the ER membrane. How is the signal sequence recognized? What is necessary for the attachment? In order to be able to answer these questions, Graham Warren and I at the European Molecular Biology Laboratory in Heidelberg split microsomes of the pancreas into soluble and membrane-integrated components. By withdrawing or adding the soluble components, we found that a soluble, cytosolic factor is necessary for the transport. Later work by Peter Walter in Blobel's laboratory identified this factor as the signal recognition particle, SRP: signal recognition particle. In my working group at EMBL, we found a second important component in the membrane, the so-called docking protein, also known as the SRP receptor. If we treated the membranes with low concentrations of a protease, they were no longer able to transport proteins. A protein on the membrane that was essential for transport had apparently been destroyed by the protease. David Meyer, then a postdoc in my laboratory, identified and characterized the protein in the membranes. He was able to show that it is necessary for the SRP-ribosome complex to "dock" to the membrane. SRP consists of a ribonucleic acid - 7S RNA - and six different proteins. It can be viewed as an adapter between the signal sequence of a growing polypeptide chain and the membrane of the endoplasmic reticulum. When about 60 amino acids of a secretory protein have been synthesized, SRP binds to the signal sequence and forms a stable complex. The 54,000 dalton protein - SRP54 - establishes contact with the signal sequence. It is more or less the postman who reads the zip code. It consists of a signal sequence binding domain, an energy-consuming switch element - the cell thus pays for the transport - and a binding site for the SRP receptor.
As long as there is no contact with the membrane of the endoplasmic reticulum, the synthesis of the protein to be transported remains interrupted. In this way the cell ensures that all secretory proteins are actually synthesized on the membrane through which they are then transported. Only when contact with the SRP receptor has been made is the protein further synthesized and simultaneously transported through the membrane.
Three switching elements, which are dependent on the energy supplier guanine triphosphate, GTP, regulate the exact sequence of the transport; one in the signal sequence binding protein SRP54 and one each in the a and b subunits of the SRP receptor in the ER membrane. The switches are in the "on" position when GTP is bound and in the "off" position after a phosphate residue has been split off. It is not yet known which individual transport steps are regulated by the switching elements. We know, however, that both the SRP receptor in the ER membrane and GTP are required for the release of the signal sequence from the SRP.
After the receptor has released the signal sequence, the growing protein chain penetrates the membrane. Since this process only takes place during the synthesis of a protein, it is difficult to clarify whether the transport occurs through a kind of protein channel in the membrane or directly through the lipids that make up the membrane. While we postulated transport through a protein channel for the signal hypothesis, there were other groups who favored direct, protein-independent transport. In order to analyze the chemical environment of the growing protein chain during the "D" broadcast, we cross-linked the protein chain with the components surrounding it and characterized the resulting complexes. To do this, we incorporated a chemically modified amino acid at certain points in a signal sequence. The modification consists of a chemically very reactive group that, when activated by light, forms a firm (covalent) bond with neighboring molecules. In collaboration with the working groups of Tom Rapoport at the Max Delbrück Center for Molecular Medicine, Berlin, and Joseph Brunner, ETH Zurich, we have succeeded in identifying a central protein of the translocation point in the endoplasmic reticulum. The "Sec61" sits in the membrane of the endoplasmic reticulum and is part of a larger complex in which, in addition to the components that are involved in the actual translocation, also Proteins that are responsible for the modification of t transported proteins are responsible, for example for the cleavage of the signal sequence and the attachment of sugar molecules.
Signal sequences of secretory proteins from bacteria and animals are very similar in their essential properties - they all have a hydrophobic region - and some of them can even function in the other system. That is, the code for the addresses, the "zip code", is largely the same in all living systems. The same applies to the receptor components that "read" the signal. We have also found a signal-recognizing particle, SRP, in bacteria, although not as elaborately built as its animal counterpart, but equipped with a small ribonucleic acid and a signal-sequence-recognizing protein. Joen Luirink, EMBL, was able to show that this particle specifically recognizes signal sequences of growing polypeptide chains. We have identified a possible receptor for bacterial SRP, the "FtsY protein", which is very similar to the animal SRP receptor. Bacteria and animal cells also have parts of the translocation channel in the membrane in common. The Sec61 protein, which was originally identified in yeast cells by genetic selection, is homologous to the "SecY protein" of the bacterium Escherichia Coli.
The general transport mechanism, the cell's "delivery system", is therefore largely the same in bacterial and animal cells: the proteins need a signal sequence for transport. This is recognized by receptors and leads to docking with the membrane. The actual transport takes place through a protein channel with the help of helper proteins on the inside and outside of the membrane.
Prof. Dr. Bernhard Dobberstein
Center for Molecular Biology Heidelberg of the University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg,
Telephone (06221) 56 68 20
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