Session 4: Vesicle Trafficking
Transcript of Part 2: Genes and Proteins Required for Secretion
00:00:07.22 Hello. 00:00:08.22 My name is Randy Schekman. 00:00:10.00 I'm at the University of California, Berkeley, in the Department of Molecular and Cell Biology. 00:00:16.26 This is the second of three lectures on the theme of how cells export proteins. 00:00:23.13 In my first lecture, I described the history of the subject and how the pioneers of 00:00:29.05 cell biology were able to understand the structure of membranes, and how membranes within 00:00:35.13 eukaryotic cells relate to one another and convey protein molecules that are encapsulated for export 00:00:43.01 outside of the cell. 00:00:45.00 In this lecture, I'm going to describe the work that started in my laboratory in the mid-1970s 00:00:51.06 to try to understand the mechanism of this process in a simple eukaryotic organism, 00:00:59.00 baker's yeast. 00:01:00.00 So, let's drill down and see what yeast cells do to export protein molecules. 00:01:06.16 First, let's have a look at a... kind of a unique angle, that depicts the process of 00:01:13.26 membrane fusion, at the very end of the process of secretion and growth of the bud surrounding 00:01:23.14 a yeast cell. 00:01:24.14 So, this is an image taken through a plane of the plasma membrane bilayer. 00:01:30.26 The cell is frozen and then a... basically, a small hammer is used to crack the cell. 00:01:38.01 And a split is obtained right through the middle of the bilayer. 00:01:43.15 And this very special image shows a bulge. 00:01:47.11 You see this bulge on the outside of the yeast cell. 00:01:51.04 This is the bud portion of the cell that I talked about last time, where cells are growing 00:01:59.02 and exporting protein molecules. 00:02:01.16 And then, enriched within this bulge or the cell bud, you see small dimples, depressions, 00:02:09.22 little craters in the membrane, that likely depict the nascent membrane fusion events 00:02:19.11 where a secretory granule had just happened to fuse with the plasma membrane. 00:02:25.19 And what you can see is, in the depression, in the dimple, we are likely imaging the molecules 00:02:34.27 that have... were encapsulated within these vesicles, but which are now being shipped 00:02:39.08 outside of the cell. 00:02:40.13 So, it's a very special image that shows that these events of membrane fusion are 00:02:46.09 likely restricted to the bud portion of a dividing cell. 00:02:50.00 So, I told you at the end of my last lecture that this process, we believe, in yeast cells 00:02:58.04 would be responsible not only for secretion, but also that these little nascent fusion events 00:03:04.11 would, step-by-step, in an iterative process, allow the membrane that surrounds the bud 00:03:11.02 to grow, to enlarge. 00:03:12.25 And therefore, if this process of fusion were intimately linked to the growth of the cell, 00:03:19.18 it would be essential for the genes responsible for this pathway to be there for the cell to grow. 00:03:28.06 In other words, if these genes were crippled by mutation, by a chemical mutation, 00:03:32.20 the cell would die. 00:03:35.02 Well, in 1976 and 1977, I had the great fortune... good fortune to have a brilliant first-year 00:03:42.15 graduate student join my lab, by the name of Peter Novick. 00:03:46.02 Here is Peter in the laboratory, busy pipetting away. 00:03:51.03 Note, this is a typical image from the 1970s. 00:03:55.00 We all had long hair back then. 00:03:56.19 And I too had long hair. 00:03:58.02 Peter has now gone on to a very successful career of his own, continuing to study 00:04:05.07 this process in yeast. 00:04:06.19 He is now, as fate would have it, the George Palade Chair of Cell Biology 00:04:12.04 at the University of California at San Diego. 00:04:14.22 So, Peter and I devised a procedure to isolate temperature-sensitive lethal mutations of yeast, 00:04:23.26 specifically focusing on those that caused secreted proteins to accumulate 00:04:31.23 inside of the cell. 00:04:33.03 Now, we were doing this, of course, in the context of the University of California, Berkeley, 00:04:39.01 the birthplace of the Free Speech Movement and the home of student protests. 00:04:43.24 And so, of course, here we were, in the 1970s, proposing to kill living organisms. 00:04:50.22 And naturally, this engendered a certain amount of criticism, indeed, even protest 00:04:55.23 against our work. 00:04:56.23 Here you see one such protest, "End the torture in the labs", "Yeast have feelings too". 00:05:03.23 We had to work hard to convince the experimental subjects committee at Berkeley that 00:05:10.02 yeast are not sentient beings, they approved our work, and we have since killed trillions of yeast cells 00:05:15.06 with no evidence of any torture in the labs. 00:05:19.10 Well, in 1978, after Peter obtained the first such temperature-sensitive lethal mutation 00:05:29.03 that caused secretory proteins to accumulate inside the cell, just by chance George Palade, 00:05:35.08 who I described in detail in my last lecture visited, UC Berkeley for two honorific lectures. 00:05:41.24 And I had the pleasure of telling him about our effort. 00:05:44.27 But, more importantly, Peter joined a group of other graduate students to organize a dinner 00:05:51.00 that evening. 00:05:52.00 It was May of 1978. 00:05:53.23 And at the dinner, he was able to tell Dr. Palade about his work and the new evidence 00:05:59.10 that he had of a mutation that blocked secretion. 00:06:01.28 And Palade naturally was quite interested and suggested that Peter have a closer look 00:06:08.07 by thin-section electron microscopy, the technique that I told you about last time, that Palade 00:06:13.25 used to such great effect in understanding the organization of eukaryotic cells. 00:06:19.17 We very quickly processed this first secretion mutant for electron microscopy. 00:06:24.22 And one of the great memories of my career came when, in the summer of 1978, Peter, 00:06:30.28 in the basement of our biochemistry building, called me excitedly down to the electron microscope 00:06:37.26 to examine images such as you see here. 00:06:41.21 In contrast to the image that I showed at the end of my last lecture, where one sees 00:06:47.14 just a few small vesicles in the bud portion of a dividing cell, in this mutant, 00:06:54.17 which we called sec-1, short for secretion-defective mutant number 1, the cell continues to make 00:07:01.16 mature secretory vesicles. 00:07:04.03 But, instead of a few in a bud portion of the cell, the cell now fills its entire cytoplasmic volume 00:07:12.23 with thousands of such vesicles. 00:07:15.24 They have nowhere to go because this gene, the sec-1 gene, encodes a protein that is 00:07:24.14 required for the granule to be attached... to become attached to the plasma membrane 00:07:32.07 of the cell. 00:07:33.07 And in the absence of that gene, in the absence of that functional protein, the vesicle has 00:07:37.27 nowhere to go, so it continues to be made, to fill the entire cytoplasmic volume. 00:07:43.20 We now know that this gene is evolutionarily conserved. 00:07:48.03 It is present in all eukaryotic cells, wherever a vesicle has to dock and fuse with a target membrane. 00:07:55.10 In fact, we even know, in the brain, in the nerve terminal, as I described in my last 00:07:59.02 lecture, that the synaptic vesicles responsible for fusion and secretion of neurotransmitters 00:08:06.22 rely crucially on a neuronal equivalent of the sec-1 gene product to organize the 00:08:14.18 fusion of a synaptic vesicle with the presynaptic membrane. 00:08:17.17 So, a billion years of evolution, and this pathway, which was... which evolved in microorganisms, 00:08:26.07 has been passed on over the eons, to be used, in molecular terms, in very similar ways by... 00:08:33.21 by human beings. 00:08:36.20 Well, this was of course a very exciting event, to see this first secretion mutant. 00:08:42.00 We knew, on the basis of how we had discovered this mutation, that there must be many more 00:08:46.21 genes to be found. 00:08:48.10 And so Peter devised another really simple and elegant way to isolate more such mutations. 00:08:55.13 He observed in the light microscope that this mutant, sec-1, when the cells are warmed to 00:09:02.09 37 degrees centigrade, continue to make these vesicles 00:09:05.07 -- indeed, they continue to make all of their macromolecules -- 00:09:08.07 but they don't get any bigger. 00:09:09.16 They don't enlarge. 00:09:10.25 They must be making more mass, probably squeezing out water. 00:09:15.20 And then they probably become more dense, more... more compact, more material 00:09:23.25 within a confined volume. 00:09:25.13 Indeed, he did the following experiment, which Illustrated that property of these cells. 00:09:32.05 He took a mixture of 99% wild type yeast cells and 1% sec-1 mutant cells, and he mixed these two. 00:09:42.28 And he incubated the mixture at 37 degrees centigrade. 00:09:45.05 And he applied the sample to the top of a tube that forms a gradient, 00:09:50.09 that would allow cells to be separated according to their buoyant density. 00:09:54.01 And he found conditions that allowed all of the normal, secretion-normal cells, to be... 00:10:01.04 to be retained at the top of this tube, at a lowboy density, whereas all of the 00:10:06.24 sec-1 mutant cells, having a higher buoyant density, sedimented to form a pellet at the bottom 00:10:11.10 of the tube. 00:10:12.10 So, he could effect a complete separation of these two populations of cells. 00:10:17.02 This is a biochemist's idea of how to select mutants. 00:10:20.00 And so what we did was we took a yeast culture, we exposed it to chemical mutagen, grew the 00:10:25.26 cells for a while, incubated the cells at a... at a restrictive growth temperature, 00:10:30.21 repeated this centrifugation step, punctured a hole in the bottom of the tube, and collected 00:10:36.01 the densest 1% of cells, plated them out on petri plates, and looked among those cells 00:10:41.15 for those that were temperature-sensitive in their ability to form colonies. 00:10:46.05 And then, among those, Peter found several hundred more mutations that defined 23 different genes, 00:10:55.00 each of which is required to produce a protein that is uniquely important 00:11:03.11 at a different point in the process of protein secretion. 00:11:07.00 He found 10 genes, mutations in 10 genes, that looked just like sec-1 in accumulating 00:11:13.14 small vesicles. 00:11:14.24 But these are different genes, which means that they are... there are at least 10 different proteins. 00:11:19.09 We now know there many more, but at least 10 different proteins that are required to 00:11:22.20 take those mature vesicles and deliver them into a bud to fuse with the plasma membrane. 00:11:28.16 He found two genes that appeared very different in the electron microscope. 00:11:33.03 Here, for example, is one such mutant. 00:11:36.04 This one is called sec-7. 00:11:37.08 And in this cell, when it's warmed to a high temperature, the cell accumulates a structure, 00:11:44.05 rarely seen... rarely if ever seen in a normal yeast cell, that looks just like the 00:11:49.03 stack of membranes that I showed you in my last lecture, discovered in nerve cells 00:11:54.15 in the 19th century, called the Golgi apparatus. 00:11:57.12 Sure enough, this structure can be seen, highly exaggerated in this mutant, because in this 00:12:04.04 mutant proteins are manufactured, delivered to the Golgi apparatus, but, because of a 00:12:11.07 defect in the sec-7 gene, they can't leave the Golgi apparatus. 00:12:15.08 So, the Golgi continues to build up to form this enormous exaggerated structure in the cell. 00:12:22.07 Now, when these cells are cooled back to room temperature, the mutant sec-7 protein is 00:12:31.15 restored to activity and the Golgi decomposes, and the proteins that have accumulated 00:12:38.22 within this structure now can be secreted to the cell surface in vesicles. 00:12:43.11 Another structure that was seen in nine mutations in the original collection caused a defect 00:12:50.20 in traffic of proteins out of the endoplasmic reticulum. 00:12:55.04 So, in this case, these mutations caused proteins to build up in the first station in this pathway. 00:13:02.01 And as a result, this organelle becomes much more elaborate, highly involved in the cell. 00:13:07.27 The nucleus, the membrane that surrounds the nucleus, the nuclear envelope, is distorted, 00:13:13.12 much wider than it is in a normal yeast cell, all because these proteins build up in the structure. 00:13:19.06 And as before, when the cells are cooled back to room temperature, the mutant protein refold 00:13:25.19 and proteins can the ER and progress as normally through the secretory pathway. 00:13:31.00 Now, this... after we had collected these mutants and published the work, we recognized 00:13:36.26 that although we had many genes there was one target that we were particularly interested in 00:13:40.27 that seemed to evade our ability to ob... to define mutations. 00:13:45.27 And that was in the machinery that Palade and Blobel, as I described in my last lecture, 00:13:55.02 showed to be required for the very first step, where secretory proteins move through a channel 00:13:59.26 in the ER membrane. 00:14:01.11 We had no such mutations and we wondered why. 00:14:04.01 And then another... as chance would have it, another brilliant graduate student, 00:14:08.26 by the name of Ray Deshaies, joined the group. 00:14:11.02 Here is Ray at a special celebration of the lab. 00:14:14.19 I'll tell you about Ray's work. 00:14:16.08 His wife, Linda Silveira, also a graduate student in the lab, worked on another project. 00:14:21.23 And Linda Hicke, another graduate student in the lab, whose work I'll describe later 00:14:26.04 in this half-hour section. 00:14:28.03 But let me tell you about Ray's idea and how he was able to conceive of a very special, 00:14:34.12 simple genetic means to identify the channel protein in the ER responsible for protein 00:14:42.13 translocation from the cytoplasm. 00:14:45.20 Here's the idea for Ray's selection, really a very simple one. 00:14:49.22 We know that most cytoplasmic proteins that act, for instance, as enzymes, remain in the cytoplasm, 00:14:57.16 where they have access to their chemical substrates. 00:15:01.15 For example, the last step in the biosynthesis of histidine is achieved by a cytoplasmic 00:15:08.24 enzyme called histidinol dehydrogenase, which takes histidinol and converts it to histidine, 00:15:15.11 which is an amino acid that's used in protein biosynthesis. 00:15:18.07 Now, we also know, from experiments that we did in yeast and which John Beckwith and 00:15:25.10 Tom Silhavy did in E coli, that if you take the gene that encodes a cytoplasmic protein, 00:15:32.18 an enzyme, and you fuse to the 5' end of that gene the sequence for a signal peptide, 00:15:43.18 a signal that would be responsible for secretion, as I described in my previous lecture, 00:15:49.19 you create a hybrid protein that now, in yeast, can be ina... inappropriately translocated 00:15:59.08 into the lumen of the ER. 00:16:01.28 In this case, by attaching a signal peptide to the N-terminus of histidinol dehydrogenase, 00:16:08.22 this enzyme is now sequestered in the ER, where it will no longer have access to 00:16:15.19 its hydrophilic substrate, histidinol. 00:16:18.21 And as a result, if this cell can't make histidine, it can't grow, unless histidine is provided 00:16:27.14 in the growth medium. 00:16:29.28 If you grow the cells on a minimal medium without histidinol, in this case... without... 00:16:36.20 without histidine, they simply won't grow. 00:16:39.01 Well, that is a perfect setup for a genetic selection, because it allows you to expose 00:16:44.24 this cell to a chemical mutagen and to look for mutations that may, for example, 00:16:52.25 cripple the machinery through which this fusion protein would be transported. 00:16:59.03 And possibly allow enough of the fusion protein to remain behind in the cytoplasm to catalyze 00:17:06.23 the conversion of histidinol to histidine, and thus allow the cell to grow. 00:17:12.07 So, to repeat, the cell contains a fusion protein that misappropriates histidinol dehydrogenase 00:17:20.16 to the lumen of the ER. 00:17:22.20 You expose that cell to a chemical that causes mutations. 00:17:26.13 And you look for cells that can now grow in the absence of histidine, in the presence 00:17:31.20 of histidinol, by virtue of the fact that the mut... that the mutation in the cell 00:17:37.16 has crippled the machinery. 00:17:39.04 Now, these mutations, of course, would kill the cell, because, as I already illustrated, 00:17:46.04 mutations that block secretion kill the cell. 00:17:48.18 So, we looked for temperature-sensitive mutations that allow the mutant protein to misbehave 00:17:57.13 just enough to leave some histidinol dehydrogenase in the cytoplasm, but mutations that 00:18:04.27 when warmed to a fully restrictive temperature, 37 degrees, kill the mutant protein completely 00:18:11.27 and prevent the cell from growing under any circumstances. 00:18:16.04 That was the crucial experiment that Deshaies did that allowed us to discover several genes, 00:18:24.04 the first of which, called sec-61, encodes a membrane protein that threads through 00:18:31.15 the ER bilayer ten times. 00:18:34.11 And which we now know is the channel in the ER, not only in yeast but in all eukaryotes, 00:18:41.01 through which polypeptides progress. 00:18:43.17 Well, let me summarize not only that but all of the work that I've described until now in yeast, 00:18:49.28 in the form of a simple cartoon that depicts the stages in the pathway 00:18:55.17 through which secretory proteins progress. 00:18:58.04 This is very much like what Dr. Palade was able to demonstrate in the mid-1970s, 00:19:05.01 but with the added bonus that each of these stages illustrated in this cartoon now can... 00:19:11.20 is populated with genes that are required at each of these steps along the pathway. 00:19:19.22 This pathway is evolutionarily conserved. 00:19:22.00 All of the genes that I've described are found in human cells. 00:19:25.25 And as a result of this discovery, it became feasible to use yeast cells as a 00:19:33.23 production vehicle for the synthesis and secretion of clinically useful quantities of human proteins. 00:19:41.11 And so, in the early 1980s, biotech companies were able to harness yeast cells by introducing genes, 00:19:48.12 such as the gene for human insulin or the gene for the hepatitis B virus surface protein, 00:19:55.12 and use yeast cells, then, to produce quantities. 00:20:00.01 For instance, one third of the world's supply of recombinant human insulin is made by secretion 00:20:04.24 in yeast. 00:20:05.28 Or, all of the hepatitis B vaccine that's made in the world, that's used for vaccination purposes, 00:20:12.22 is made by producing vesicles in yeast cells that house the hepatitis B virus protein, 00:20:19.22 which can then stimulate the immune system. 00:20:22.10 So, this was a practical benefit of the basic science that we and others in my laboratory performed. 00:20:29.12 But we were interested in understanding mechanism. 00:20:33.04 And though we had the tools available to define the genes, by itself the existence of 00:20:40.26 these genes, in the early 1980s, didn't tell us what we really wanted to know, which was, 00:20:45.22 how does this process work? 00:20:47.17 What do these genes encode? 00:20:49.04 What do the molecules, the protein molecules encoded by these genes do to manufacture vesicles 00:20:55.01 that allow cells to grow and secrete proteins? 00:20:59.05 And for this, I'm going to introduce two new observations that allowed us to make progress. 00:21:04.14 The first was a closer look at this first step in the pathway, performed by 00:21:10.17 a wonderful postdoctoral fellow in the lab at the time by the name of Chris Kaiser, who had a close... 00:21:15.08 a very much closer look by microscopy and genetics at the genes required to convey proteins 00:21:20.26 between the ER and Golgi apparatus. 00:21:24.10 Here's a summary of what Chris Kaiser discovered. 00:21:28.07 He found that among the set of genes required for the movement of proteins between these 00:21:34.06 two organelles there are two subsets which show extensive genetic interactions, 00:21:42.10 among each group separately. 00:21:44.25 And which, in the first instance, are required 00:21:48.11 to produce vesicles by budding from the endoplasmic reticulum. 00:21:53.18 And in the second instance, to take these vesicles and to deliver them, 00:21:58.22 by membrane fusion, to the Golgi apparatus. 00:22:02.11 Now, we cloned and sequenced these genes. 00:22:06.00 And very interestingly, two of the genes required for the fusion of these vesicles at the Golgi apparatus 00:22:13.13 turn out to be the yeast equivalents of two proteins that James Rothman and his 00:22:20.11 colleagues had purified from mammalian cells that seemed to be responsible for the fusion 00:22:26.20 of vesicles in the mammalian Golgi apparatus. 00:22:30.25 He purified two proteins, called NSF and alpha-SNAP, which turned out to be the human or mammalian 00:22:40.08 equivalents of the yeast genes sec-18 and sec-17. 00:22:44.28 So that, by the end of the 1980s, we were able to appreciate not only the evolutionary conservation 00:22:50.08 of this pathway but, at the detailed molecular, mechanistic level, genes in yeast 00:22:57.04 had the equivalent function to proteins obtained from mammalian cells. 00:23:02.21 Now, in the final part of this lecture, I want to take a step back to tell you about 00:23:09.19 a historical precedent for how you can use this kind of genetics to bootstrap an understanding 00:23:17.01 of biochemical molecular mechanism. 00:23:19.28 So, now we're going to take a step back, 20 years, to a crucial landmark experiment conducted 00:23:27.20 by two investigators at Caltech in 1965. 00:23:32.23 Here they are. 00:23:33.23 These are Bob Edgar, a bacterial virus geneticist, 00:23:40.15 and his young protege, a new assistant professor at Caltech, by the name of William Wood. 00:23:46.26 Edgar was a classic geneticist. 00:23:49.27 He used the bacteriophage T4 to understand the genes that are required for the production 00:23:57.01 of infectious virion particles. 00:23:59.08 He discovered that mutations in these genes blocked the production of virus particles 00:24:04.16 and caused infected E coli cells to accumulate incomplete virions. 00:24:11.02 But he had no idea what these gene products may do to promote the assembly of the virus. 00:24:17.14 Bill Wood had come as a trained biochemist... trained in the laboratory of Paul Berg 00:24:23.26 at Stanford University, so he was well versed in biochemical analysis. 00:24:28.19 And he saw the great advantage of what Edgar had done to conceive of a strategy, 00:24:33.25 that I'll show you now, that allowed this team to develop a cell-free reaction that reproduced the production of 00:24:42.15 virion particles in the test tube, employing the genes that Edgar had discovered by 00:24:48.03 his genetic approach. 00:24:49.23 Here's the basic experiment. 00:24:53.02 One starts with bacteria that are infected with two different virus mutants, 00:25:01.08 each by themselves incapable of making infectious virions. 00:25:06.02 Now, if these cells had been infected with the two viruses together, genetic complementation 00:25:13.09 inside of the infected cell would have allowed one defective genome to complement the other 00:25:19.21 to produce infectious viruses. 00:25:22.16 But if the mutant viruses were used to infect two different populations of bacteria, 00:25:29.08 nothing would happen. 00:25:30.12 What Wood properly recognized was that if these two different populations were broken 00:25:38.13 and the cytoplasm fractions from each were mixed in the test tube there may be 00:25:47.11 a form of biochemical complementation that would allow each virion to provide the missing component 00:25:56.10 for a completion of virus assembly in the test tube. 00:26:00.04 Indeed, that is what's happened. 00:26:02.02 And the data shown on the right shows a beautiful result, where, at the outset of the experiment, 00:26:09.06 very few if any infectious virus particles are detected. 00:26:12.28 But, as the two mutant samples are incubated in the test tube together, three logs of infectious 00:26:21.07 virion particles are produced during a 30 or 40-minute incubation. 00:26:26.17 This is a result that warms the heart of any biochemist. 00:26:30.19 And provided a historical precedent that we in my laboratory could use to try to 00:26:38.21 identify biochemical entities associated with defective gene products. 00:26:43.08 We struggled for some years to achieve such a reaction. 00:26:46.26 But eventually another brilliant graduate student by the name of David Baker, 00:26:51.02 who has gone on to his very successful own career, joined the lab, and within a very short period of time 00:26:58.08 David had devised a very simple way of breaking open yeast cells that would 00:27:04.11 allow communication, vesicular communication, between the ER and Golgi apparatus to be reproduced 00:27:12.11 in the test tube. 00:27:14.10 His work was followed by the efforts of two graduate students, whose work I'd like to 00:27:20.04 tell you about now. 00:27:21.19 The first was Linda Hicke, whose picture I showed you a few minutes ago. 00:27:25.20 Linda was a graduate student in the lab working on a gene that we now know to be required 00:27:31.03 to form vesicles that bud from the ER. 00:27:34.15 And she used the system that David Baker had developed to do a variation on the Wood and Edgar 00:27:42.07 experiment that I just told you about. 00:27:44.10 And I want to show you the data for that, because it's an experiment that 00:27:48.01 still warms my heart to this day. 00:27:50.12 She took the following combinations of extracts of cells. 00:27:55.18 The dark column shows a sample that was incubated with membranes that by themselves showed 00:28:05.17 no transport as defined by the Baker assay, but could be restored to activity when the sample 00:28:12.28 was incubated at a permissive temperature for the cell-free reaction, in this case, 00:28:18.25 15 degrees. 00:28:20.01 This dark line is of cytosol taken from a sec23 mutant, a mutant that would be defective 00:28:28.20 if the cell had been incubated at 37 degrees, but would be nearly normal if the cell 00:28:33.15 was incubated at a low temperature. 00:28:35.17 Indeed, this cell-free reaction was quite active at the temperature 15 degrees. 00:28:42.05 She also prepared cytosolic proteins from a cell that has a wild type copy of sec23. 00:28:49.17 And similarly, this mixture of wild-type cytosol and membranes produced transport activity 00:28:56.11 as measured in the Baker assay. 00:28:58.27 Now, crucially, as independent samples were incubated at slightly higher temperatures, 00:29:07.13 from 25 to 30 degrees, which we found to be a restrictive temperature for our 00:29:14.15 biochemical assay, the activity for transport with cytosol carrying the wild-type SEC23 protein 00:29:23.14 was more or less intact. 00:29:25.11 But importantly, the activity associated with cytosol carrying the mutant SEC23 protein 00:29:32.22 was down quite markedly. 00:29:35.12 And clearly, then, a temperature-sensitive traffic reaction indicated that this assay, 00:29:43.07 the Baker assay, faithfully reproduced the pathway that we had deduced on the basis of 00:29:51.21 genetic analysis, confirming that this was a functional assay that would allow us 00:29:58.04 to purify these proteins. 00:29:59.20 Now, this observation was further simplified by a very important contribution of 00:30:06.01 another terrific graduate student by the name of Michael Rexach. 00:30:09.06 Rexach observed that, in the course of the cell-free reaction that Baker had devised, 00:30:16.20 membranes in the lysate, specifically ER membranes in this gentle lysate, remained very large, 00:30:25.04 and could be sedimented to the bottom of a centrifuge tube with a very low-speed centrifugation. 00:30:31.21 Her further observed that if these large membranes were incubated with wild-type cytosolic proteins, 00:30:40.02 during the course of an incubation at 30 degrees, small vesicles formed that could not be sedimented 00:30:48.19 to form a pellet at the bottom of a tube. 00:30:51.21 And instead would have to be sedimented only after a very high-speed centrifugation. 00:30:57.21 So, a simple differential centrifugation, of the sort that I described at 00:31:03.22 the outset of my last lecture, was sufficient to separate vesicles that bud from the ER in vitro 00:31:12.00 from the ER membranes. 00:31:14.06 Further, Rexach showed that the mutants, such as sec23, are defective in the production 00:31:21.24 of these small vesicles. 00:31:23.15 Well, this then allowed us to begin to fractionate all of the proteins that we knew to be 00:31:32.22 required for vesicle budding, those genes that Chris Kaiser had described that are responsible 00:31:38.15 for vesicle budding in vivo turn out also to be required for vesicle budding in vitro. 00:31:44.02 And as a result, we were able to discover that these genes have a unique function to 00:31:51.08 assemble on the surface of the ER membrane, to form a bud that pinches from the ER membrane. 00:32:00.09 In order to visualize this process, we developed a collaboration with 00:32:03.19 one of the great morphologists in the world, today, 00:32:06.13 a man by the name of Lelio Orci, shown here in his office in Geneva, 00:32:11.09 with whom I had the pleasure of collaborating for over 20 years on experiments of the following sort. 00:32:18.06 We purified all of the proteins necessary to bud vesicles from the ER, the genes that 00:32:24.12 we had already described by genetic analysis and their protein products, and we took these 00:32:29.25 purified proteins and we mixed them with ER membranes, we sedimented away the ER membranes 00:32:35.00 at low speed, and obtained a high speed pellet fraction. 00:32:38.10 And with the guidance of Lelio Orci, we were able to visualize the vesicles that formed 00:32:44.07 in the test tube, and were amazed to see that a uniform population of about 80-nanometer vesicles 00:32:53.26 were produced in the test tube, each of which is coated by a... what appeared, 00:33:02.08 at least initially, to be a fuzzy electron-dense coat, consisting of the proteins that we added 00:33:09.12 to perform the budding reaction. 00:33:11.11 Now, we now know, and I'll summarize in the next two slides, that the proteins that 00:33:16.25 do this assemble stepwise to produce a bud, to pinch the bud to form a vesicle, 00:33:23.06 and to capture membrane and secreted proteins 00:33:27.11 that are designed to be conveyed from the ER to the Golgi apparatus. 00:33:32.07 So, let me show you a summary slide and then a higher-resolution image of how these proteins work 00:33:37.23 in my last two slides of this section. 00:33:41.10 First, this is a cartoon, summarizing a great deal of work over a period of many years, 00:33:46.20 that describes how this process works. 00:33:48.17 And let me just summarize it for you in just a little detail. 00:33:52.18 We know that this process of budding begins with a small GTP-binding protein called Sar1 00:34:02.20 that acquires GTP by interacting with a membrane protein called Sec12, where it lodges 00:34:10.09 into the ER membrane to begin to deform that membrane to form a bud. 00:34:16.04 We then know that two proteins, in the form of a heterodimer of two Sec gene products, 00:34:22.17 Sec23 and Sec24, assemble onto the dimple that's formed by Sar1, and begin to 00:34:31.18 sample different membrane proteins for capture into a nascent bud. 00:34:37.01 They recognize... specifically, the Sec24 molecule recognizes sequences on membrane proteins 00:34:45.06 that are signals for traffic out of the ER. 00:34:49.12 And complexes are formed that then are clustered together through the intervention of 00:34:56.25 the outer layer of this coat, the Sec31/Sec13 complex, which literally envelops the membrane, 00:35:05.22 in the form of a scaffold, to sculpt the bud and to pinch it, collecting, as it does so, 00:35:13.02 not only the inner layer of the coat but also cargo molecules that are designed to be transported, 00:35:19.05 while excluding proteins in the ER membrane that are designed to remain behind and 00:35:24.12 not to be transported. 00:35:25.18 Now, we now know... through the work of other laboratories that have taken these proteins 00:35:30.18 and developed atomic-resolution crystal structures, we know a great deal about how the molecules 00:35:38.19 of this COPII coat cooperate to form this bud. 00:35:43.15 And let me summarize that in my last slide for this part. 00:35:47.05 This is a lower-resolution image. 00:35:49.17 We now have much higher atomic-resolution images. 00:35:53.02 The coat consists of two layers. 00:35:55.23 There's an inner layer of proteins, consisting of the GTP binding protein Sar1 and its partners 00:36:02.24 Sec23 and 24, responsible for tagging cargo molecules designed to be transported. 00:36:10.06 And then, an outer layer, this outer layer of two other Sec, proteins Sec13 and 31, 00:36:17.04 that form a scaffold. 00:36:18.19 Indeed, this scaffold, as was first described by William Balch and his colleagues in La Jolla, 00:36:24.06 this scaffold has the unusual ability to self-assemble into a regular polyhedron, 00:36:32.03 a cube octahedral structure with squares and triangular facets that forms the kind of exoskeleton 00:36:41.10 that surrounds the membrane, capturing cargo molecules for budding from the ER membrane. 00:36:48.08 Well, we know a lot about these, not only in yeast but also in mammalian cells. 00:36:53.11 We even know that some human genetic diseases are the result of mutations in different subunits 00:36:59.08 of this COPII coat, once again confirming the evolutionary conservation of this pathway. 00:37:06.06 And emphasizing something that I feel very strongly with... about, and which I'll 00:37:09.23 leave you with for this part. 00:37:12.02 Which is that the pursuit of science for its own curiosity-driven thirst for understanding, 00:37:21.19 inevitably, when one discovers things of a fundamental nature such as I've described... 00:37:28.01 inevitably has practical application, in this case, in the biotechnology industry, 00:37:34.25 and even in understanding at a fundamental level how human diseases may evolve. 00:37:39.12 I'll leave you with that for this part of the lecture series. 00:37:44.06 And we'll start in a few minutes with my third lecture, which will describe a process 00:37:50.14 that probably doesn't happen in yeast, but which also involves the capture, in this case, 00:37:56.21 of RNA molecules into vesicles that may be transported within the human body and promote not only 00:38:04.05 normal development but also may be subverted in human disease. 00:38:08.15 Thank you.