However, some proteins are retained in the ER, by a means that is still not understood, and others are returned to ER after they have entered the Golgi apparatus. The signal for the return pathway involves the sequence KDEL (lysine, aspartate, glutamate and leucine), as I mentioned previously. There is KDEL receptor protein in the Golgi that recognizes and binds to this sequence and then returns in a vesicle to the ER. This implies that there is a specific retrieval pathway for some proteins that have been carried to the Golgi. There are other proteins in the ER for which there is no evidence that they have ever left and it is believed that some "retention" signal allows them to remain there. However, the existence and nature of this signal remain to be identified.
Most proteins made in the ER, however, leave it and move into the Golgi apparatus. The Golgi apparatus seems to serve several functions. One is that it's a place where proteins undergo extensive covalent modification, especially modification of their oligosaccharide side chains. That is, there is in the Golgi a series of enzymes that are capable of cutting some sugars off the oligosaccharide core and attaching others. Usually the glucose that was attached in the ER is removed by "glucosidases" in the ER. But the protein with an N-linked core oligosaccharide composed of mannose and N-acetylglucosamine is then transferred to the Golgi, where it is further processed by removal of some sugars and addition of others. Some of this cutting and adding involves the same sugars that are attached in the ER--mannose and N-acetylglucosamine. However, some reactions occur in the Golgi that don't happen in the ER. For example, sugars such as sialic acid (N-acetylneuraminic acid) and galactose are only attached to the oligosaccharide side chains of proteins in the Golgi. This implies that certain enzymes are only present in the Golgi (and others only present in the ER), and there is evidence to support this. Moreover, the Golgi enzymes are not randomly distributed throughout the Golgi but are usually restricted to one of the three regions--cis, medial, and trans--Golgi. In fact, the enzymes are localized in the Golgi in the order in which they act on the oligosaccharide side chains of the proteins. (Fib. 17-38) So the enzyme catalyzing the first reaction (mannosidase 1) is in the cis Golgi, the enzyme catalyzing the next in the medial Golgi and so forth. That means that not only are the Golgi and ER different, but different parts of the Golgi itself have different components. That in turn implies that there must be some mechanism to target various components to different areas of the Golgi, but how this occurs is not yet understood.
Proteins and membrane fragments don't just hop by themselves from the ER to the cis Golgi and from one Golgi compartment to the next. They are transported by budding off of membrane sacs, called vesicles--little spherical structures that contain a membranous coat and a fluid-filled lumen. These transport vesicles move from one compartment to another by budding off one and fusing with the next. How this happens is a very active area of research, and we'll discuss what is known about it in a few days.
I said that the Golgi apparatus had two main functions. One is modification of glycoproteins. Though this is clearly something to which the cell devotes a lot of energy, it is still not clear what the advantage of having this array of oligosaccharides on proteins is. In some cases, proteins that are blocked from being glycosylated seem to function perfectly well, suggesting that glycosylation isn't essential for initial folding, but often these unglycosylated proteins are unstable--that is, they're more readily degraded than the glycosylated form of the protein. For other proteins there is evidence that glycosylation is essential for proper folding of the protein into its active conformation or stabilization and inhibition of degradation.
The other main function of the Golgi apparatus is sorting. The Golgi is the final common pathway of membrane, some organelle, and secretory proteins . As they leave the trans Golgi, they are sorted and sent to different destinations. Again there seems to be a default pathway--namely transport of vesicles to and fusion with the plasma membrane. However, there are also alternative pathways by which particular components of the Golgi apparatus are pulled out of the default pathway and sent to other destinations in the cell, including lysosomes and endoplasmic reticulum. (How can one tell whether a protein in the ER has passed through the Golgi and returned?)
So lysosomes are little digestive centers in the cells, eating lots of stuff from both inside and outside the cell. In order for them to function, they must obtain both the digestive enzymes that degrade macromolecules and some food. So there must be some mechanisms to get both the enzymes and the food into the lysosome.
First let's consider how the enzymes get there. Consider the following situation. A patient comes to you with what you recognize is I-cell disease. This person's cells have lysosomes that are full of undigested material, and that lack acid hydrolases. On the other hand, their blood is full of these digestive enzymes, which normally are not released from cells in appreciable amounts. You further know that this disease runs in families and is a recessive Mendelian disorder. That is, a person needs to inherit a defective copy of a single gene from both parents in order to show the disease. What hypothesis can you generate to account for this patient's symptoms--i.e. what is the cellular basis of I-cell disease? You have to account for why their lysosomes are defective and why their lysosomal enzymes are extracellular. And then you need to devise experiments to decide whether your explanation is correct. What experiments would you do to test this hypothesis?
It was in fact in studying this kind of lysosomal storage disease that biologists discovered what the signal was for directing proteins into the lysosome. People with I-cell disease had lysosomal enzymes in their bloodstream that lacked an uncommon sugar that was always found on the oligosaccharide chains of enzymes in the lysosomes of normal cells--mannose-6-phosphate. There is now considerable evidence that mannose-6-P is the signal for sorting in the trans Golgi that directs a protein away from the default pathway and into vesicles that travel to and merge with the lysosomes.
If membrane vesicles are constantly moving from the Golgi to the lysosomes, then lysosomes should get bigger and bigger as they accumulate more and more membrane, and perhaps the Golgi would get smaller. But this doesn't happen. Why not? There must be some way for membrane to return from the lysosome to the Golgi so that there is a steady state, with the lysosome neither gaining nor losing membrane over time. It's not clear how this reciprocal traffic is regulated, but it's not only important in reclaiming membrane, but in reclaiming specific proteins. For example, the receptor protein that binds the mannose-6-P tagged acid hydrolases would quickly get used up if it moved only one way from Golgi to lysosome, leaving no receptor in the Golgi to pick up new enzymes. So the receptor needs to get back to the Golgi. It's still not clearly understood how receptor knows to go from Golgi to lysosome, when it has the enzyme bound, and then back to the Golgi when it's empty, but it is known what the signal for dumping the acid hydrolase with mannose-6-P is--it's the acid pH of a pre-lysosomal compartment (called the late endosome for reasons that will become clear later). Somehow the acidity reduces the affinity between the receptor and the ligand so that the enzyme dissociates from the receptor once it reaches late endosome and the receptor can be recycled back to the Golgi.
That's basically what we know about how the enzymes get into the lysosomes. How does the stuff they eat get there? There seem to be three mechanisms for delivering food to lysosomes. (Fig. 5-44) One involves the degradation of endogenous organelles--i.e., those that normally live inside the cells, like mitochondria. It's thought that this pathway involves the ER membrane somehow enveloping the organelle that will be degraded and then carrying it to the lysosome, but the details of this process are not well understood at present. Much better understood are the pathways that involve degradation of exogenous materials--those that normally exist outside the cell in the blood and other bodily fluids. These pathways involve endocytosis, the process by which the cell engulfs a portion of its own plasma membrane. Historically, cell biologists have distinguished two kinds of endocytosis--one is phagocytosis, which means cell eating and involves the uptake of big particles from outside the cell like bacteria or other cells of the body. The other form, which used to be called pinocytosis, or cell drinking, involves retrieval of membrane by the cell without any visible particles attached although it is now abundantly clear that many molecules are taken up in this way. Pinocytosis is usually called endocytosisthese days, and phagocytosis is considered as a process by itself. They differ in one important way. Phagocytosis is a triggered process--that is, it only happens if something tells the cell to do it. Endocytosis (pinocytosis) is constitutive--it goes on all the time.
What triggers phagocytosis is something big binding to specific receptor proteins in the plasma membrane of the cell. When that happens the cell begins an uptake process in which the attached particle is fairly quickly surrounded by the cell's membrane which is pinched off and then internalized, ending up eventually in the lysosomes.
The best studied of all the pathways to the lysosome is endocytosis, and the reason it's the best understood is that it is very important in uptake of crucial cellular components like iron . Another is that it's medically important. Consider the following situation. A family comes to you in which there is a very high incidence of heart disease; people often die of heart attacks in their 20s. When you do blood tests of the affected family members you find that they have astronomical levels of cholesterol in the bloodstream, and that this cholesterol is forming large deposits on the walls of their arteries, choking off the flow of blood and causing atherosclerosis. You know that cholesterol does not float around free in the bloodstream, because it's an extremely hydrophobic molecule. Rather it forms a complex of hundreds of cholesterol and other lipid molecules with a large protein--this complex is called the low density lipoprotein (LDL) particle (Fig. 17-45), and this particle is what your cholesterol tests are detecting in the bloodstream of these patients. The disease also appears to be a Mendelian recessive disease, and it's called familial hypercholesterolemia. What's wrong with them? Any guesses?
What's wrong is that their cells can't retrieve LDL from the bloodstream. LDL is a way for cholesterol that's in your diet to get from your digestive system to the cells that use it to build their membranes. But if their cells can't take up LDL, it accumulates in the bloodstream and causes disease. There people have defects in the uptake pathway and in studying what the nature of this defect was, Brown and Goldstein discovered much of the endocytic process, and they won the Nobel Prize for their work. They asked what is the normal pathway for LDL uptake and what is wrong in the patients? To answer this question they tagged LDL and then examined how it was processed by cells in tissue culture. They compared the processing by cells from normal and from affected individuals.
In the family members who did not have hypercholesterolemia they found the following model. (Fig. 17-46) LDL bound to receptors that were present uniformly on the surface of the cell. Very quickly after the LDL bound however, the receptors attached to them redistributed on the cell surface. They were no longer randomly distributed but rather bound in small depressions or pits in the membrane that were lines on the cytoplasmic side with a meshwork of a protein that was purified and named clathrin. This could be seen in the EM. These depressions with clathrin coats are called coated pits, and the LDL receptors quickly get stuck in these pits when they bind LDL. The pits are the precursor to a vesicle that buds off from the membrane, called a coated vesicle. It's a sphere of plasma membrane with a basket of clathrin around it. The vesicle moves into the cell, sheds its coat, which returns to the plasma membrane, and then quickly fuses with a membranous structure that is near the outer surface of the membrane. This organelle was discovered as a result of these studies and is called the early endosome (i..e., the body that occurs early in the endocytic pathway). The early endosome appears to fuse with a sorting vesicle (aka late endosome, which has a low pH (around pH 5). This causes the LDL to dissociate from its receptor, so that now the LDL is free in the lumen of the early endosome. By a process that's not entirely worked out, the LDL receptors can be recycled back to the plasma membrane in vesicles that pinch off the late endosome, while other vesicles bud off the endosome and move deeper into the cell where they fuse with vesicles from the Golgi that contain degradative enzymes. When the two kinds of vesicles fuse, the LDL is for the first time exposed to degradative enzymes; this new structure is called a lysosome. As the LDL particle is broken down, its protein and fatty acids are degraded by acid hydrolases, but cholesterol, which has no bonds formed by dehydration, is not affected. Rather it is released by the cytosol where it can be carried by transport proteins to the smooth ER and incorporated into new membrane. Some of the cholesterol also binds to the enzymes that normally synthesize cholesterol and inhibit them. That is, high levels of cholesterol in the diet normally shut down the synthesis of cholesterol by the cell. In some cases, the synthesis of the enzymes that make cholesterol is also turned off; i.e. transcription of their genes is also inhibited by cholesterol.
This endocytosis pathway picks up lots of stuff, including an iron-transporting protein, called transferrin (Fig. 17-48). A variety of other proteins, including small signaling proteins, called cytokines, that bind to receptors in the cell membrane, are taken up in this way. When protein uptake requires that the protein binds to a specific cell-surface receptor, the process has been called receptor-mediated endocytosis.
But to come back to the question I posed earlier: what's wrong with the folks who have familial hypercholesterolemia? What kind of mutation could mess up this pathway? Most mutations affect the receptors for LDL in the plasma membrane. Some cause no production of any receptor protein--what kinds of mutations would do that? (Some possibilities include either nonsense mutations that create a truncated protein or a mutation that alters the signal sequence so the protein is not longer inserted into the membrane--how would you distinguish them?). Some make a receptor protein that is correctly placed in the membrane but that don't bind the LDL. What sort of mutation would do that? (The mutation is presumably in the extracellular part of the protein that contains the LDL binding site.) Some have a normal ability to bind LDL, but they don't aggregate in coated pits after they do, so they just sit on the cell membrane instead of being internalized. What kind of mutation could cause that effect?
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