The ER is an essential organelle in the cells because it's the place where membrane components are made and where most of the components that reside in the interiors of the cell's compartments are made. It's also the place where secreted proteins are synthesized. As you know, the ER is divided into smooth ER, so-called because of its appearance in electron micrographs, and rough ER, which appears irregular because it is studded with ribosomes. Rough ER is far more prevalent in most cells, and in some cells like the pancreas and neurons it fills the cell’s interior. As you might deduce from the fact that the rough ER contains ribosomes, it is a place of very active protein synthesis.
The importance of the ER was first worked out in a series of Nobel prizewinning experiments by George Palade and his coworkers. They were interested in the process by which cells made secretory proteins. They studied cells from the pancreas because it was known that the pancreas made and secreted a large number of enzymes into the intestines that were important in digestion (Fig. 5-47; 5-48). The method that they used is called a pulse-chase experiment. The idea is that a pulse of radioactive amino acids is delivered to the cells (in this case by injection into a rabbit's bloodstream). For a brief period the proteins that are made become radioactive, then one "chases" the radioactive amino acid with unlabelled amino acids. In this way one gets a brief period of synthesis of radioactive proteins. The procedure is to find out where the radioactive proteins first appear in the cells, which is presumably where they are being made, and then where they go. Then, you can identify the sites of radioactivity by autoradiography, in which the cell is coated with a radiation sensitive photographic emulsion. When the radioactive particles and radiation from the labeled protein molecules interact with the emulsion, they cause silver grains to precipitate (it's silver that gives photos their black appearance). These precipitated silver grains can be seen in the electron microscope. Since the radioactivity usually hits nearby silver grains, you see the grains only in the regions where the cell contains radioactivity.
Back to Palade's work. He injected radioactive amino acids into pancreas, and looked to see where radioactive protein appeared and when. He found the following result. (Show overhead of autorad.) He got a similar result when he fractionated the cell components. When he plotted the amount of radioactivity in various regions of the cell versus time after the pulse of radioactive aas, he got the following graph. What interpretation can you give to these results?
So these experiments provided the first evidence that secreted proteins were synthesized in the rough ER and that they could move through a series of cellular compartments before they were secreted. This raised a number of questions. First how did the proteins get into the rough ER? Second what were the mechanisms that allowed them to move from compartment to compartment? Palade's work was done in the 1950s and '60s and people have been busy ever since trying to answer those questions.
I'd first like to tackle the question of how proteins get to the ER. As you'll see, or you already know, both secretory proteins like digestive enzymes and membrane proteins get made there, in essentially the same way. People realized early on that it was going to be difficult to get a protein into or through a membrane once it had been made in an aqueous environment, because such a protein will have to fold up one way in water and a different way in lipid. What's more it's pretty clear from the kinds of experiments that we've just been discussing that membrane and secreted proteins were being made in the rough ER, not on free ribosomes in the cytosol. So that suggested that membrane and secretory proteins were pushed into or through the membrane as they were being made, and then the question became--how did membrane proteins know that they had to be synthesized on ribosomes in the ER and not on free ribosomes? Discuss some hypotheses and some tests. One possibility was that there was a special class of ribosomes associated with the ER and that mRNAs for membrane proteins had some particular affinity for those. How to test this idea? Another possibility was proposed by Blobel and Sabatini, who called their idea the signal hypothesis. They proposed that there was nothing special about the ribosomes or the mRNA, but that the membrane protein itself had an affinity for the ER. The original hypothesis was that the first part (N-terminus) of any membrane or secretory protein contains a sequence of amino acids that binds to some receptor in the ER membrane. This binding does two things. It attaches the mRNA, ribosome, and nascent (which literally means "being born" or "emerging") protein to the ER and it begins to thread the protein through the membrane. They imagined that the synthesis of the protein on the ribosome could push the protein all the way into or through the ER membrane, and then this signal sequence is usually cleaved off by an proteolytic enzyme called a signal peptidase.
Their ability to test their hypothesis experimentally depended on their ability to break apart cells and separate the different parts from each other, a procedure call cell fractionation. This usually depends on the technique of differential centrifugation. As explained in Lodish et al., (pp 154-155), it's possible to homogenize cells and to separate the components using the techniques of differential centrifugation and density centrifugation. In differential centrifugation you centrifuge cell homogenates at sequentially higher and higher speeds, thereby pelleting smaller and smaller components. (Fig. 5-23). If you homogenize a cell and spin at low speed, the nuclei--which are the largest organelles--are spun down into the pellet, but all the other organelles stay in solution. So you can pour off the supernatant which contains the other organelles, and keep the pellet which is essentially pure nuclei. If you then centrifuge the supernatant at higher speeds larger organelles--the mitochondria--are pelleted. If you use very high speeds, you bring down into the pellet small fragments of the er and Golgi apparatus, which are called microsomes. This process is called differential centrifugation, because at different speeds, you obtain different materials in the pellet. You can thus fractionate the cell's organelles.
Some organelles can't be separated in this way, such as smooth and rough ER (microsomes), but they can be separated if they are layered onto a centrifuge tube containing a solution of sucrose of increasing density from top to bottom. (Fig. 5-24). In this circumstance, the cell organelles move down the tube in the centrifuge until they reach the region of sucrose that has the same density as they do, where they reach equilibrium and stop (they float up if they go into a region of higher density, and spin down from a region of lower density.) This is called equilibrium density gradient centrifugation and can be used to further fractionate parts of the cell that can't be separated by differential centrifugation.
First Blobel and then many others found that when secretory proteins are made in vitro with just ribosomes, mRNA, amino acids and tRNA, the protein that is synthesized is almost always slightly bigger than the normal, functional protein. (How would one determine this?) If they added microsomes --i.e., purified ER--then the proteins were the normal size. This suggested that microsomes could nibble off part of the protein. (Fig. 17-15) We know believe that the signal hypothesis is essentially correct, although the intervening 20 years have made clear that there's more than this simple model suggests going on. Much of the knowledge is the result of work by Blobel and his collaborators, and it's a virtual certainty that he'll get the Nobel Prize for this work (I said in 1998; he received the Nobel Prize for Medicine or Physiology in 1999). (Take a look at the quicktime movie--protein secretion.)
Look at Fig. 17-16. Observe roles of SRP--6 proteins and an RNA that can be washed off the microsome by high salt, , hydrophobic N-terminal signal sequence, SRP receptor, ribosome receptor, protein translocation pore (translocon), signal peptidase, and hsc70 or BiP chaperone proteins. (Fig. 17-16 shows the situation worked out in yeast cells; other eukaryotes are slightly different. Also check out thequicktime movie "Synthesis of secreted protein").
The SRP consists of 6 proteins, along with a small RNA molecule (that probably binds to the rRNA in the ribosome). One of the proteins, P54, has a group of methionines, with fairly hydrophobic side chains that binds to the signal sequence of the nascent protein (Fig. 17-17). The binding of the SRP to a nascent peptide prevents further elongation of the protein. Once the SRP binds to the SRP receptor complex, and the ribosome transferred to the "translocon", the SRP is ejected from the complex, allowing protein synthesis to resume. The components of the translocon were identified primarily using genetic methods in yeast, where many mutants defective in secretion have been identified. Some fail to take up secretory proteins into the ER (so the proteins remain in the cytosol), and a subclass of these have mutations in two proteins (sec61p) and Translocating-chain associated membrane protein = TRAM.
As you surely know by now, the movement of proteins through membranes is an active process requiring the cell to cough up some free energy. Most of the free energy necessary for insertion of proteins into the ER seems to come from GTP hydrolysis (you may recall that most of the free energy for protein synthesis is also derived from GTP hydrolysis). Both the SRP and SRP receptor are associated with GTP, which is broken down to GDP during the binding of the SRP-ribosome to the SRP receptor, and insertion of the protein into the translocon (Fig. 17-20). Hydrolysis of GTP is in fact probably required for dissociation of the SRP. In some cells additional free energy is provided by the chaperone protein in the lumen of the ER, which binds the emerging protein as it passes through the translocon, and prevents it from folding prematurely, at the expense of ATP hydrolysis. In mammalian cells, the chaperone protein is called BiP (binding protein) and it doesn't seem to hydrolyze ATP, so GTP may be the sole source of free energy for translocation in mammalian cells.
No comments:
Post a Comment