Scientist Stories: Randy Schekman, Genetic Basis of Vesicle Transport

Thank you all for coming to this rather different lecture. I've, of course, visited Hopkins before for the purpose of seminars, but this is going to be a more of a, let's say, retrospective where I'm going to review some of the history of cell biology that led to the work of my students and I at the Department of initially Biochemistry, then Molecular and Cell Biology at UC Berkeley. But first, a little bit about my own history. Although I was born in Minnesota, I really grew up in California. I'm a California guy. You can tell by my tan and my blonde, bleach blonde hair. My interest in science emerged at a very early age. I had a toy microscope given to me by my parents, probably for a birthday. And in Orange County where I grew up back then, there actually were lots of farm fields and not too many subdivisions. And there was a creek near my home where one day I went and collected a jar of pond scum And I came back and I put a drop of this on a glass slide under this microscope. And I was captivated from that moment. To see the world, the microbial world emerge even from this plastic lens was somehow, you know, reverberated with me. And I was so excited that evening that I told my parents about it. My father was rather skeptical that I could see anything like that in a toy. So I became actually quite offended at his dismissing— dismissive remarks. So I vowed to save money to buy myself a student professional microscope. So I mowed lawns, I babysat, I delivered newspapers, and I was slowly building up to my goal of $100, putting it in an envelope in the closet in my bedroom when I found that my mother kept borrowing the money to buy groceries. One Saturday I was so angry that I bicycled from my chore of mowing a lawn to the police station and I told the duty officer that I was running away from home because my mother was stealing my money and I couldn't get my microscope. So, they called my father in and there were some words behind a closed door and he came out looking rather severe. And— but I learned a valuable lesson that afternoon because we drove right from the police station to a pawn shop in Long Beach— so origins and influences— to a pawn shop in Long Beach where he put down the remaining money and for $100 I bought this Bausch and Lomb student microscope, which was my pride and joy through the remainder of what was then called junior high school and high school. And every year I mounted an effort using this microscope to look at microorganisms of one sort or another for my annual science fair project. But of course, life moves on and I went to college. I went to UCLA. They had better microscopes there, so this got put away in the garage, but we'll come back to this later. I developed— when I was in college, I developed an interest in chromosome replication, initially virus chromosome replication. But really more broadly in understanding cellular mechanisms, how processes work within cells. Initially it was with DNA replication and I had the great good fortune to be admitted to Stanford, to the biochemistry department and then to the laboratory of Arthur Kornberg, one of the dominant biochemists of the late 20th century. I learned from Kornberg not only really how to work hard to satisfy him, but also more importantly, how to look at a complicated problem and to take it apart piece by piece, putting it back together in vitro to reconstitute a complex reaction. This was for me a really formative experience that I attempted to replicate as my career developed. I wanted to do something different though for my future studies, so I moved to the study of biological membranes and was a postdoctoral fellow in the lab of S.J. Singer at UC San Diego just after his very famous publication of the fluid mosaic model of membrane structure. I learned from Singer a little bit about how biological membranes were put together, but I learned a great deal also from the literature, principally of the masterful work of George Palade and Keith Porter, who in the 1960s and early 1970s were able to appreciate how membranes within a cell communicate with each other through a flow of vesicular traffic. This is the problem, the complexity of the problem is illustrated by an image a thin section image of a secretory cell that's engaged in the production, in this case, of insulin. You'll be familiar with this cell. It's the beta cell of the pancreas. And you know that this cell is devoted to the production of insulin. You see insulin here housed within granules that are stored and discharged by membrane fusion when the cell is stimulated to secrete insulin. But these organelles and the insulin itself come from earlier membranes in what constitutes the secretory pathway. In each case, the organelle that surrounds secretory proteins that move step by step through the cell consists of a double-track biological membrane whose overall organization is well described by the fluid mosaic model of membrane structure. You all know about this. The bilayer consists of two leaflets of phospholipids, each surface of which is presented at a hydrophilic interface with a core of fatty acids that constitute the hydrophobic interior. And each membrane is distinguished by its content of unique membrane proteins. Permeases in the membrane that allow the transit of small molecules or receptor molecules, receptor proteins that receive signals and transmit information by a process of signal transduction. So all the membranes in the cell are organized roughly in this way and they communicate with one another somehow. But it took the genius of George Palade to chart the pathway step by step. I never had the opportunity to work directly with Pilati, but I got to know him early in my career, even as early as when I was a graduate student in Kornberg's lab because the two were good friends. Indeed, Kornberg did a series of little mini-sabbaticals, including a several-month stretch at Rockefeller University working directly with David Sabatini, who was a colleague of Pilati's. So they developed a deep appreciation for each other's scholarship. Palade had, in the preceding decades, mastered the use of the electron microscope, which you see here him standing— sitting behind later in his career as a professor at UC San Diego. He mastered this tool along with Keith Porter to be able not only to visualize organelles, but as you'll see in a couple of minutes, to be able to stage the traffic pathway by a very elegant experiment, which now seems old-fashioned and very complicated compared to what we can do, but at the time it was really revolutionary. The first organelle that he characterized, both morphologically and biochemically, is the endoplasmic reticulum. You know this as the platform on which ribosomes rest to transmit by a process of vectorial translocation newly synthesized proteins that penetrate through a membrane, the ER membrane, into the luminal space of the organelle, representing a kind of a network of canals within the cell that collect all molecules that will, in this case, eventually be secreted. In this case, a pancreatic exocrine cell is professionally devoted to the production of secretory proteins. Almost 80% of its energy is consumed by protein synthesis of secretory proteins. Indeed, before then, Palade had actually discovered the ribosome. Simply by inspecting thin sections of cells, he saw these regular stained dots, and he was able to show by fractionation that these represented the seat of protein biosynthesis in cells. He was then able to isolate the ER membranes with bound ribosomes and show that this so-called rough microsome preparation could be incubated in buffer with protein synthesis goodies. And the ribosome caught in the act of producing a secretory protein would continue that process, resulting in the deposition of the completed chain in the lumen of this isolated vesicle, demonstrating that there must be a channel in that membrane. Subsequently, his protégé, Günter Blobel, refined that reaction, was able to reconstitute the entire event to demonstrate that secretory proteins have an N-terminal signal that directs them likely to a site on the ER membrane. And for that, Palade was awarded the Nobel Prize in 1998, some years after Palade did in 1974. Well, the next station that Palade highlighted was an organelle called the Golgi apparatus that had already been described many years earlier, indeed at the end of the 19th century by an Italian cytologist by the name of Camillo Golgi who found in mixing various stains that a particular combination highlighted this organelle rather than others in nerve cells. And its function was entirely obscure, but it acquired his name, the Golgi apparatus, subsequently. And for nearly 60 years, no one really had any idea about what this may do. Indeed, Most people thought it was some kind of a staining artifact. Well, it took Palade, as you'll see in a moment, to figure out that this is a station intermediate between the ER and secretory vesicles in which secretory materials are processed and packaged for delivery to the cell surface. The final event that Palade was able to visualize, a very important terminal event, where the granule housing, in this case insulin, reaches up and touches the perimeter of the cell. The membrane surrounding the granule becomes very closely opposed to the inner surface of the plasma membrane. And at a key point, these membranes merge by a process called membrane fusion where topologically what was inside of the vesicle becomes ejected to the cell exterior. All the while retaining the impermeable barrier constituted by the plasma membrane. So, you can export things outside of the cell without breaching the barrier of the plasma membrane. This event is crucial. It's often regulated. It's complex. But at this point, phenomenologically, all we knew was this event occurred. We also knew that the event occurred quite widely distributed in a variety of tissues serving a number of functions. For example, an image of the neuromuscular junction, in this case the frog neuromuscular junction, shows the accumulation of what in this case constitute smaller so-called synaptic vesicles that carry neurotransmitters for discharge at the presynaptic membrane. When an action potential reaches the presynaptic space, calcium flows in through a calcium— a voltage-gated calcium channel, that triggers the action of a very special membrane protein called synaptotagmin that was discovered by Thomas Sudhoff, my fellow Laureate. It's controlled by calcium binding to cause the SNARE proteins, most carefully studied by Jim Rothman, that engage both the pre- and postsynaptic— the synaptic and presynaptic membrane to process by fusion, to merge by fusion, resulting in the discharge of neurotransmitters into the cleft between the nerve cell, in this case a muscle cell. Subsequent to Palladi's work, Heuser and Reese were able to carefully synchronize this process by a very careful timed discharge with a resolution of as little as 5—within 5 milliseconds of generating a local action potential. They could see the Synaptic vesicles merge by fusion to discharge content to the synaptic cleft. This process, let me emphasize again, is repeated all over the cell and in many different cells and serves many essential purposes. Well, putting it all together, what Pallotti did was he figured out a way to pulse radiolabel tissue with carbon-14 amino acids and then impose a chase with cold amino acids, taking samples at each time point for chemical fixation, dehydration, embedment into a plastic resin, followed by thin sections which were then applied to a very thin photographic emulsion and put aside in a drawer for months, allowing radioactive decays to be registered by the deposition of a grain track, which could then months later be visualized and superimposed on the on the thin section of the tissue sample. And he could show, as is in cartoon form shown over here, that material moves in this pulse chase step by step from the ER through a vesicle intermediate to the Golgi apparatus to granules and eventually to the cell surface. Now, I'll never forget that Palade, after, directly after receiving his Nobel Prize, attended the American Society for Cell Biology meeting in 1974, which just by chance was held in San Diego where I had just recently arrived as a postdoctoral fellow just one month earlier. Palade was asked to give a special lecture, basically a reprise of his Nobel lecture, and he recounted these really beautiful experiments and I learned more about the the remarkable man. It was such an event with a devoted audience that he received a standing ovation after this lecture. Although I appreciated the power of microscopy and organelle fractionation as he had developed it, having been trained as a biochemist in Kornberg's lab, I was left with more questions than he was able to answer. Specifically, what cellular genes and proteins organize this process? Surely a complex series of events like this would be catalyzed by enzymes, structural proteins, all kinds of things which at the time were completely unknown. In 1974, not a single molecule, not a single protein molecule, certainly not a gene, had been associated with any step in this pathway. We had no idea about the nature of the channel through which secretory proteins pass into the lumen of the ER. We had no idea about the machinery necessary for production of a vesicle by some budding process. And very specifically, we knew nothing about the catalyst that may allow membranes to touch each other and then merge by membrane fusion. Surely this would be a fascinating complex set of interactions that could be studied by some kind of molecular approach. However, in 1974, the tools available for the dissection of any complex process such as this in a mammalian tissue were simply not there. One could not use traditional genetics and biochemistry of that sort was very difficult with such tissues. So when I began my own career in 1976 at UC Berkeley, I resolved to take a turn to a different direction to rely on a microorganism where one could use the traditional tools of genetics and biochemistry with precision to identify genes and proteins. And I was motivated not only by my experience in Kornberg's lab but by the emerging dissection of the cell division cycle in Lee Hartwell's lab at the University of Washington. He had isolated a series of mutations in genes that are required sequentially for the progression of a cell through its cycle. And he could identify these genes because he assumed that such genes would be essential for cell division, and thus he was really driven to using a very classical technique, the isolation of temperature-sensitive conditional lethal mutants that block at individual steps. Well, I'm sure most of you know about yeast, so I'll just be very brief in my introductory remarks about it. It grows by a budding mechanism. The cell sends out a small bud when nutrient conditions are right. The bud grows in preference to the mother cell during the 90 minutes to 2 hours of the cell division cycle. And just a simple inspection of such cells, such as mitotic grow on the surface of a grape, gives one the impression that surface area in the yeast cell division cycle must be added by a sequential process of enlargement of the bud membrane, the membrane that surrounds the cytoplasm and is itself protected by a porous cell wall barrier. Indeed, if you look inside of a yeast cell, you see some evidence of organelles that may feed growth of the bud membrane. Yeast cells have an overwhelming impression of ribosomes. The cytoplasm is densely packed with ribosomes, but there are some organelles characteristic of the secretory pathway. There's a vacuole that is the yeast equivalent of the lysosome. There are tubules or sheets of ER membrane, in this case largely opposed to the inner surface of the plasma membrane. But Peter Novik, one of my first graduate students, and I were focused on a cluster of small vesicles that congregate under the bud portion of a dividing cell. And we made, we speculated that these vesicles would not only be a means of conveying secretory proteins into the growing cell wall, but importantly that the membrane surrounding the vesicle would constitute a building block for the growth of the plasma membrane of the cell. So a process, by a process of secretion, one could in a quantal fashion grow the plasma membrane step by step. If so, then prediction as with the cell division cycle is that the genes required for the biosynthesis, the movement, the docking, and the fusion of these vesicles would be essential genes and thus we would be obliged to look for temperature-sensitive lethal mutations which when incubated, when cells, mutant cells were incubated at 37 degrees, would accumulate secretory proteins inside the cell instead of being secreted into the cell wall. Well, all of this early work was conducted by, you know, I was just, I've just been so fortunate, a wonderful graduate student, Peter Novick, shown here in a rare moment of away from active pipetting. You see here sort of a cultural characteristic of the early 1970s, this long hair. Any of you who know Peter now would be amazed by this picture. He and I have much less hair than we used to. Well, so— but I have to tell you this was done in the context of UC Berkeley, the birthplace of the protest, student protest movement. And whenever you propose to kill a living being at a place like Berkeley,, it's going to engender some controversy. Indeed, there was some protesting about our work. "End the torture in the labs." "Yeast have feelings too." We had to get approval from the animal subjects committee. I persuaded them that yeast are not sentient beings and we have since killed trillions of yeast cells. So we set about to look for temperature-sensitive lethal mutants. There was a kind of a, you know, hit-or-miss style because neither Novick nor I had any genetics experience. Neither of us had worked on yeast, so it was fun at first, but after a while we decided to survey just a random collection of temperature-sensitive colonies. Cells were mutagenized, plated on rich medium, and simply replica plated to find colonies that were temperature sensitive for growth in the hope that this would be a complex enough pathway that we would just by chance hit on a gene that happened to be involved in this as opposed to, you know, one of the many other functions that would be essential for cell division in yeast. So he found such a mutant early in 1978 and he showed that at the high temperature the cells stopped growing and they build up two different secretory enzymes reversibly in an intracellular pool that can be released when cells are returned to a permissive temperature. And just by chance, right then, May of 1978, George Palade visited Berkeley for two special lectures that he delivered. It was wonderful to meet him again and to share with him what we were doing on secretion in yeast. He was surprised to learn that yeast cells secrete glycoproteins. But more importantly, that evening the students organized a dinner with Dr. Palade and Peter had the opportunity to show him his results with this mutant that accumulated secretory proteins. And Palade naturally said, "Well, have you looked inside the cell? Does it— what does it look like by thin-section microscopy?" We hadn't done that yet, but we were, of course, motivated to do it very quickly. And within short order, one of the great experiences of my life, in the early summer of 1978, I was busy writing a grant in my office when Peter called me excitedly from the electron microscope room in the basement of our building, and I ran down to see one of the following images. This is a mutant, sec1 mutant cell. After incubating the culture at a high temperature, 37 degrees, for a couple of hours, the cell accumulates thousands of mature secretory vesicles that fill the entire cytoplasmic compartment. Not just the very small number that we saw in an earlier image. These cells become chock-full of such vesicles because it turns out the Sec1 protein, a membrane, a soluble protein, is required to organize the SNARE proteins that engage vesicles with the plasma membrane. Indeed, this protein, the Sec1 protein, is found throughout the cell at different stations where membrane fusion must be executed. And it— one finds it even at a nerve terminal. There's a neuronal form of Sec1 that engages neuronal snares to promote synaptic vesicle fusion at the nerve terminal. Well, this was very exciting, but again, it was sort of from a random mutagenesis, and so we thought there are surely— there are many more genes. We need a way of finding them. And we decided at that point that it would be necessary to have some kind of a genetic selection. And so what Peter did was, on closer inspection, he realized that these cells have an unusual physical feature. As they are incubated at the restrictive temperature, they continue to accumulate mass but they do not enlarge. They don't get any bigger. Quite unlike the temperature-sensitive cell cycle mutants of Hartwell, which continued to form unusual shapes. And continue to grow. These cells freeze in place and they become phase refractile. So Peter guessed that these cells would become— would increase their buoyant density. Their cytoplasmic density must increase. And this then allowed a very simple procedure to separate mutant cells from wild-type cells on a density gradient. This is a biochemist's idea of genetic selection. So what Peter then did, another really cool experiment, he took the mutant cell and he introduced a gene that would allow us to document the mutation on a colony just by a histochemical stain. And then he mixed a small population of mutant cells with wild-type cells that didn't— weren't so marked. And he incubated the mixture at the high temperature and then applied the cells to a density gradient that resolved cells according to their buoyant density. And he found remarkably that all of the mutant cells from this mixture were now present at a pellet just near the bottom of the tube, whereas all of the great excess of wild-type cells remained at the top of the gradient at a lower buoyant density. So a density gradient, in a way, purification of mutant secretion mutants. So over the next 18 months, there was a rush to use this procedure over and over. He isolated an additional 220 mutant-independent mutant alleles that defined, by complementation analysis, 23 different genes that intervene at several distinct stages in a linear pathway. 10 of the genes that we identified show the same phenotype as sec1. They accumulate mature secretory vesicles. So that says that last step, it has at least, and we now know more, at least 10 proteins that are involved in docking and fusion of vesicles at the bud tip. A couple of other mutations define genes required for the progression of proteins through the Golgi apparatus. Now normally in a wild-type yeast cell you never see anything like this unless it happens to be labeled with a G, but in this case you didn't even need the label because it's so obvious that the structure builds up and looks very much like the typical stack of pancakes characteristic of a mammalian Golgi apparatus. Novik could show that in this mutant secretory proteins progress into this structure, they remain there when the cells are incubated at a restrictive temperature, And when they are cooled to room temperature, the proteins progress into vesicles and out of the cell. Another phenotype that he found caused a block in traffic of proteins out of the ER. In this case, this structure, the ER becomes highly exaggerated, the lumen is distended, the nuclear envelope is highlighted much more so than normal, and one can show that secretory proteins in this mutant accumulate in this otherwise clear interior space. But when the cells are returned to the permissive temperature, the proteins progress step by step to the cell surface. Well, Novik and then subsequent several generations of students could map where these genes act by using very simple genetic epistasis and electron microscopic evaluation to produce a pathway that we appreciated in the early 1990s that looks formally the same as the pathway that Pilati had described, but with the bonus, the considerable, I would say, bonus of a lot of genes that define each of the steps. For example, Ray Deshaies, a subsequent really brilliant student in the lab, devised a different kind of procedure to isolate mutations that define the subunits of the channel in the ER membrane through which secretory proteins progress. This is a channel that is fundamentally conserved in all organisms from bacteria and archaea through all of the eukaryotes. We characterized, as you see in a moment, a set of genes that are required for the biosynthesis of vesicles that bud from the ER membrane. And former postdoctoral fellows Tom Stevens and Scott Emmer in their own labs were able to define a limb, a branch point in the pathway that leads to the transport of proteins from the Golgi apparatus to the vacuole. These also are highly conserved and required for lysosomal biogenesis in mammalian cells. In his own lab initially at Yale, Peter focused on a gene called Sec4 and discovered that it encodes the first of a now very large family of so-called RAB proteins, small GTP-binding proteins whose function is to direct a vesicle to a particular target. In mammals, there are over 3 dozen of these RAB proteins indicating the variety of choices that a vesicle must make in determining its final destination. Now, at around this time or, you know, in the years around 1990, of course, it became possible to clone and sequence all of these genes by a simple cloning using complementation with a plasmid library, and some of the genes had some— had sequence that had some instructive value, but most of them did not. And even when the mammalian genomes became available and it was clear that all the Sec genes have their counterparts in higher organisms, that by itself didn't tell us anything about what most of these proteins actually do. What are their functional roles? In traffic within the cell. Now, I'd always anticipated this, and again, it was probably because of my training in Kornberg's lab that I was drawn almost immediately to attempting to reconstitute a significant aspect of the pathway in vitro, in a cell-free system with isolated membranes and cytosolic proteins, just as Jim Rothman had at the time done with lysates of mammalian cells. It was some struggle, but eventually another brilliant graduate student by the name of David Baker joined the lab, and within 2 weeks he had devised a way of gently rupturing yeast cells, preserving the interorganelle traffic of vesicles all in a cell-free system. Linda Hickey, who was a graduate student in the lab at the time, was able to show that the reaction that Baker reconstituted was a faithful replica of the cellular event because lysates prepared from mutants that block traffic in vivo do so at precisely the same step in the in vitro reaction. Further, her observation allowed us to develop a biochemical complementation assay that we could use to purify individual components from an otherwise crude extract containing many of the other Sec proteins and everything else that would be required at other stages. We could purify by biochemical complementation one thing at a time. We did so, and in 1994 we were able to show that the vesicles that are produced from the ER in vitro are uniform in size. And all contain a kind of a fuzzy coat that surrounds the vesicle. At first it seemed rather amorphous, but on close inspection, indeed in the laboratories of Jonathan Goldberg and Bill Balch and others, these proteins were all crystallized. And we now know at atomic resolution the structure of all of the subunits that constitute what we call the COP II coat responsible for vesicle formation in the ER in all organisms, in all eukaryotic organisms. Here's just one low-resolution snapshot of the layers of the coat. An inner layer of the coat consists of a small GTP-binding protein, Sar1, and two Sec proteins that gather together and seek out by diffusion recognizing cargo molecules that present a sorting signal on the cytoplasmic face of the ER. These COAT subunits bind to the signal and mark that molecule for capture into a cage structure formed by two other Sec proteins, Sec13 and Sec31, that Bill Balch showed create a regular polyhedral lattice that forms just with mixed pure proteins. This polyhedral lattice assembles about the surface of a membrane, deforming it into a bud and then by fission pinching the bud from the membrane, capturing in the vesicle membrane largely only those cargo molecules that are designed to be moved from the ER to the Golgi apparatus. So this coat achieves both vesicle morphogenesis and cargo protein sorting. Now, at around this time in the mid-1990s, I was content to continue to work on yeast. It still was, and is I believe, the most powerful eukaryotic organism for dissection of essential processes. I doubted at that time that anything interesting would turn up in the clinic with any patient with mutations in these genes, because after all, these are all single essential genes in yeast. However, that was naïve because it became clear quite quickly that the genes that are single in yeast are at least duplicated in the mammalian genome, and as a result of different patterns of tissue expression of the paralogs of these molecules, there has been unfortunately an opportunity for some genetic mischief. For instance, this gene that encodes the second copy of SAR1 is defective in patients with a disease called Anderson's disease. These patients have a defect in the packaging of the giant lipoprotein called a chylomicron into COP II vesicles that bud from the ER in enterocytes of the intestinal epithelium. And we're actively investigating why this paralog, which is very similar to the other one, should have such a dedicated role. This set of genes, the Sec24s, constitute a set of proteins required to recognize discrete cohorts of cargo molecules for packaging into a COP II vesicle. This became clear initially through a collaboration with your former colleague David Ginty, who was on the faculty here, who found in his laboratory a mouse mutant defective in neural tube closure with an early chain-terminating mutation in the mouse copy of this Sec24B. In collaboration with Ginty, Devin Jensen, a graduate student in my lab, was able to show that Sec24B is very particular about its cargo preferences, and it seems to be required for the packaging of a protein involved in the morphogenesis of the neural epithelium a protein called Vangl2. And we could show— actually the Ginty lab could show that Vangl2 in the embryo of a Sec24B essentially null allele, Vangl2 accumulated in the ER, in the neural epithelial cells. So that was a very instructive collaboration. In another collaboration with David Ginsburg and his postdoc, Xiaowei Chen, at the University of Michigan, they found that deleting this gene, Sec24a, produces what appears to be a normal mouse, but which curiously lacks or is severely reduced in the level of free cholesterol and LDL-bound cholesterol, a very interesting, possibly useful feature of this mutant. It turns out, on close inspection, that Zhao Wei Chen found that the defect in the Sec24a mutant is an inability to recognize and package a very interesting serum protein called PCSK9. Some of you may know about this molecule. It's now a very important molecule in the pharmaceutical industry because PCSK9 determines the itinerary of the LDL receptor. When the levels of PCSK9 protein are high, the LDL receptor is diverted to the lysosome and degraded,, and cells behave as though they are similar to cells from patients with a disease called familial hypercholesterolemia, and they generate a very high level of cholesterol. On the other hand, there are patients with lesions in the gene for PCSK9, and in this case the LDL receptor has a normalized itinerary. It can take care of LDL particles and can control the peripheral production of cholesterol, and these patients have an exceptionally low level of free cholesterol, and as a result, they have a low incidence of heart disease. So now, several pharmaceutical companies have developed monoclonal antibodies that neutralize the PCSK9 protein, and these— this has presented a completely orthogonal method of control of cholesterol that works— that can work synergistically with the statins that control it by blocking the action of HMG-CoA reductase. So, again, this now presents perhaps another target for drug opportunity. In the next few minutes, I'd like to describe an investigation that we conducted, once again with a former colleague of yours, Simeon Boyev, who is a pediatric geneticist here at Hopkins. Now, about over 10 years ago, Boyev called to say, all— very excitedly, that he'd, with a Saudi Arabian colleague, he'd found a group, a Bedouin family that had a rare craniofacial disorder that they were able to map to a lesion in one of two paralogs of the SEC23 gene, which is a partner of SEC24 involved in the inner layer of the coat. Mutations in this gene have a very selective defect, as you'll see, and in other work independently conducted elsewhere, mutations in its paralog copy that patients with a genetic form of anemia have been found because this paralog, turns out, is expressed preferentially in the erythroid lineage at the expense of the SEC23A, and conversely, SEC23A is expressed predominantly in cells that feed bone morphogenesis. So, patients with this disease called CLSD, are shown here. Here are two kids that are homozygous deficient in this SEC23A paralog. They have facial dysmorphism, they have— they develop cataracts, brittle bones, the fontanelle, the top of the head, never closes, but they are otherwise seemingly fairly normal, which was remarkable to us because we were able to show, and as you'll see in my last piece of data that skin cells harvested from these kids are profoundly defective in secretion. We collected a sample, a skin biopsy from one of the carriers, the mother, who had no pathology and compared it by thin-section electron microscopy to cells from one of the kids, and that is shown in this last data slide of mine. The top panel is of cells of normal fibroblasts with the rough endoplasmic reticulum. The middle panel is from the mother, a carrier, who showed again no pathology but with some apparent distortion of the ER. But skin cells, fibroblasts harvested from one of the kids, showed a profound dilation of the ER. And we showed by, in other experiments, that the granular material that accumulates within the lumen of the ER is largely collagen, which has great difficulty in secretion in these cells. So, that we think is a partial explanation for the disease. Anyway, there are many such stories that now have developed as a result of what we learned in yeast applying quite directly to issues that may affect human disease and for which our understanding of basic mechanisms may eventually have some application. Well, I was, after some years of working on this, called on to sort of summarize in bullet points the most important things that we think we've established over now nearly 40 years at Berkeley. And so I'd like to summarize the major conclusions of the work that I've described. We have demonstrated that the process of secretion is intimately linked to the process of to the growth of the plasma membrane through a series of physically and functionally linked organelles and that are obligate in the pathway. This is a step beyond what Palade's experiments could say. With the availability of the genes cloned and sequenced, we could say that the process of secretion is highly evolutionarily conserved. And in the case of the initial translocation of it, the channel is conserved from bacteria through archaea and all eukaryotes. This conservation allowed in the late 1970s when we were first getting going and making these discoveries, allowed the biotechnology industry to harness yeast as a source for production, large-scale production of clinically important proteins. And the local company, Chiron, that I consulted for found that when they expressed the hepatitis surface antigen in yeast, it caused the cells to accumulate little vesicles that were filled with this surface antigen, but obviously without the virion nucleic acid. And if any of you have been immunized for hepatitis B, you will have received a vesicle preparation from yeast into your veins. Subsequent to this, Chiron engineered the gene for human insulin and now 1/3 of the world's supply of insulin is produced by secretion in yeast. More specifically, we showed in recent years that the COP II coat is responsible both for sorting cargo molecules into transport vesicles but also for shaping these vesicles. And finally, we could explain some of the pathologies of diseases affecting COPD components because they have different patterns of tissue expression. Now let me return in the final moment to that microscope that long sat collecting dust in my— it turns out my wine room in my basement. My children were not interested in science, I may have said, and so the microscope sat there lonely until a week after I got the call about the Nobel Prize, I received an email from the Nobel Museum in Stockholm. And every year they ask laureates to donate something to the museum, a kind of an artifact from the past. So here you are. If you come to the museum, you can visit my microscope and the docent will tell you how I had to run away from home to get this, to start my career. Well, let me then finish by showing you the people in my group now who have carried the ball while I've been traveling around giving talks like this. It's a wonderful group that fortunately is very independent. I visit occasionally. I'm considered a visiting professor in my own laboratory now. But they keep the enterprise going. Now, I have two short subjects that I'd like to deal with. The first I— we thought you might like to see. When the— on the day of the Nobel Prize ceremony there are two major events. Presentation ceremony in the concert hall in Stockholm and then later that day, later in the evening, there's a huge banquet with 1,300 people all dressed up very fancy. The meals are served by a fleet of servers who descend down stairwells delivering plates simultaneously to all 1,300 people. There's dancing and singing. It's quite a lavish affair. And then at the very end, each group of laureates is asked to select one representative to make some remarks of appreciation to the group. And if all things work well, I was fortunate enough to be asked to do this for my group. [FOREIGN LANGUAGE] Physiologie et la Médecine. It is a great honor to introduce the Nobel laureate in Physiology or Medicine, Professor Randy Sheckman.

Your Majesties, Your Royal Highnesses, ladies and gentlemen, on behalf of my colleagues Jim Rothman and Tom Sudhoff, I am deeply honored and humbled by the recognition you have bestowed on us today. I view this occasion as one of the great moments of my life, one that I'm thrilled to share with my wife and children, my father and family, friends, friends, colleagues, and the students of mine who made this day possible. I wish particularly to acknowledge the Nobel Foundation for its recognition of basic science. This year's laureates in the natural sciences reflect the value of curiosity-driven inquiry, unfettered by top-down management of goals and methods. Government funding of basic research in the United States started after World War II, with a transformative report, Science: Endless Frontiers, written by Vannevar Bush, the science advisor to Presidents Roosevelt and Truman. He wrote, scientific progress on a broad front results from the free play of free intellects working on subjects of their own choice in the manner dictated by their curiosity for exploration of the unknown. Freedom of inquiry must be preserved under any plan for government support of science. That freedom of inquiry nourished the careers of today's laureates. Jim Rothman and Tom Sudhoff discovered the molecular basis of neurotransmitter release at the junction of nerve cells and between nerve and muscle cells. The molecules they probed are the targets of a potent a potent toxin produced by a poisonous bacterium that causes botulism. No one could have predicted a practical application of this knowledge. Yet judging from the many handsome, near-perfect faces here this evening, some of you may have personal experience with Botox, the commercial term of this toxin. The uses of Botox extend well beyond the cosmetic. Including the treatment of debilitating neuromuscular diseases such as afflicted the concert pianist Leon Fleischer, who after 30 years of paralysis has now been restored to the concert stage. The work in my laboratory probed the molecular basis of protein secretion in baker's yeast. We had no notion of any practical application of this work, And yet, after we learned that yeast cells use a pathway fundamentally the same as in human cells, the biotechnology industry applied this knowledge to engineer the production of commercially useful quantities of human proteins. One-third of the world's supply of recombinant human insulin is produced in yeast. Again, it is likely that some of you here tonight benefit from this discovery and technology. Many of you can recount similar stories where an investment in basic science has resulted in a direct application to medicine and technology. And yet we find a growing tendency for government to want to manage the discovery with expansive so-called strategic science initiatives at the expense of the individual creative exercise we celebrate today. Louis Pasteur recognized this tension long ago before the trend toward managed science. He wrote, "There does not exist a category of science to which one can give the name applied science. There are sciences and the application of science bound together as the fruit of the tree which bears it." And so to the memory of Alfred Nobel for his vision whose vision to recognize individual scientific achievement, and to Louis Pasteur and the beverage industry he saved. And to yet another example of yeast fermentation technology, I offer a toast to our gracious Swedish hosts.

Skål! Okay, so I want to turn to one last thing that occupies my time more, almost more than anything. I'll just spend just a few minutes on, and that is the question that concerns us all, and that is where do we seek to publish our most important work? We face, all of us, a challenge because of pressures to publish in a very small number of venues, the journals that I now refer to as the luxury journals that are very highly selective. And in doing so, you face a gauntlet of evil people, some of whom are shown here. You have to endure a review process, of course this is true at any journal, but at some of the journals they throw more of these evil people at you repeatedly until your paper is accepted. Journals such as Nature and Cell and Science have a wonderful business plan. It is their brilliant idea that if they created, if they create a club, an exclusive club that you all want to join, that you will flock to them as a feeding frenzy to try your best effort to get your work published in these venues. They sustain this feeding frenzy by amping up a phony number that everyone in this room knows about, some of you to 3 digits, a number called the impact factor. This is a number that has no meaning in the measurement of scholarship of individual achievement. Achievement. It is a measure of the number of citations in a journal over a brief window, a 2-year period. And it is a reflection of, in the case of Nature, Cell, and Science, a very small number of papers that generate a disproportionate number of citations. The impact factor, you may not know, is calculated as an average, not as it should be, as a median. And as a result, numbers like this at Nature are inflated inflated by a minority of papers that generate hundreds of citations. What are some of these papers? Well, one of them you may remember was published by Nature early last year. Two papers that claimed that adult cells could be converted into embryonic stem cells by exposure to low pH. These papers were an instant sensation. Evening news, front page of the New York Times. 1.2 million hits on the link to that paper within the first 2 months after it was published. I'm sure those papers generated an increase in impact factor in Nature, but as you know, they were wrong. People tried and failed to replicate the results around the world, and within weeks, the first author who had returned to Japan to celebration was humiliated. Her supervisor at the Reagan Institute where she was working and fellow co-author on the paper, was so humiliated that he committed suicide. This is a tragic story. Of course, there are human frailties involved here and wherever fraud is committed, as we learned about these papers, they were almost certainly fraudulent. But what you don't hear, and what I will tell you now, is that I believe that the journals bear some responsibility for creating this pressure that we all feel and that in some cases causes people to cut corners, to misrepresent their work, and in extreme cases to commit fraud in the effort to get their papers published in such journals. It takes other pathologic turns. Here's an example. You can't read this but I can assure you it's— maybe some of the Chinese near the front can read this. A document that was published 2 years ago to the community of academics of the Chinese Academy of Sciences. Basically, it's a reward notice that says that if you win the lottery and you happen to publish in Cell, Nature, or Science, irrespective of the paper, you get a personal cash reward of $33,000 for your pocket. There are some Chinese scholars who are so effective at this lottery system that over half of their income comes from these rewards. Now, I have to say, as the former editor of the PNAS, I was kind of offended that the PNAS paper was only worth $5,000, and of course they didn't even bother to list eLife here. Now, it's funny if it— it would be funny if it weren't true. This is true, and I submit to you that this is a distortion of scholarship. The use by administrators, by groups of faculty, by university officials, by publishers in all countries, even in this country, is a distortion of the meaning of scholarship. Two years ago, a group of us journal editors, including myself and Bruce Alberts, then the editor of Science, gathered in San Francisco to propose a document that I hope many of you have seen It's called the DORA, Declaration of Research Assessment. It's been posted online. It's now accumulated over 12,000 signatures. It calls for all of us to move away from the, from the really embarrassing use of this phony number as a measure of scholarship. And it calls for other metrics or other means of evaluating scholarship. For example, reading the papers. This has been signed by many journal editors. Regrettably, the editors of Nature and Cell have declined to sign this document. Well, you know, we can complain, we can rail against the system, but 4 years ago I was asked by the Howard Hughes Medical Institute, by the Max Planck Society, and the Wellcome Trust to take on a new journal, and we've created a journal that's had some success. It's called eLife. It's completely free. Funded, supported by these organizations. We have several principles. We will publish all the important papers that we review and accept. We will not target for a small number of papers. Of course, we're selective, but we don't wish to be ridiculously so. It's an online journal. It's open access. There's no reason to limit either the number of papers or the number of pages that we accept. Nature and Science, on the other hand, have a significant print copy and their policy is to limit papers in length and in number simply because of the expense of additional pages in a print run. I think this has no place in the 21st century. Now, of course, there are some people who object to our policy of using scientists as editors rather than professional editors. Sorry, Adam. But there are other objections. Some people think that impact factor is good. It turns out there's now a Twitter feed from a group that's promoting Trump as a possible replacement of Francis Collins as the director of the NIH. Shocking but true. It turns out that maybe, you know, maybe he doesn't get the Republican nomination, maybe he has another job as directing the biomedical enterprise in the country. So, this Twitter feed contained a number of interesting entries. Most interesting for me was the following huge news: we won negotiations with Macmillan. All Nature journals to be rebranded Trump. All will have impact factors of greater than 50. Suck it, Sheckman. Now, regrettably, I will not be on the stage next week. Debating Donald Trump. Instead, I'll be lecturing lectures like this. Thank you for your attention. So now we're going to start the question and answer part of the event. Ladder? Stand up. For a lot of young scientists, you do a PubMed search, you're kind of overwhelmed with thousands of hits, and I know for myself one of the screening methods I kind of used for a while was looking at Nature, Cell, and Science, those kind of journals, their associated journals first. Do you have any recommendations for how to evaluate— you'll see, you find a journal that you've never heard— and it seems really, really specific and you don't know how to trust it or not. Do you have any advice on that? Yeah. Well, I would encourage you to read more broadly and not just to focus on those journals. Some of the things they publish are correct. Why, I've even published in those journals over the years. But let me also point out that some of the greatest biochemical investigations were published in journals that you may not read anymore. Al Gilman published all of his great work in the Journal of Biological Chemistry. Avram Hershko published his seminal work on the discovery of ATP-dependent proteolysis, which led to the discovery of ubiquitin, in the BBRC, a journal that you may never even have heard of. So there are great papers out there. I'll give you another example. Last year's Nobel Prize in Chemistry, one of the winners was Eric Betzig, who now is at Genelia Pharm,— he invented super-resolution microscopy, a specific form called PALM. The key discovery, the key theoretical underpinning of that discovery was published in a journal called Optical Physics, which to this day, after the— in the last 15 years, has only accumulated 100 citations, probably most of them in the last 2 years. So I think you're missing an awful lot if you focus only on Cell, Nature, and Science, and just remember how I argue they are selecting papers. They're looking for things that they think are going to be instantly recognized to generate citations. I know they will deny this, but just look at what they— look at what they publish and look at what they don't publish and realize what you're missing if you look only at those journals.

Yes? Thanks very much for the speech. It's a great honor to hear from you. Another push lately has been for the increasing need of reproducibility in science, as well as for post-hoc review processes. And I'm wondering what your opinions are on attempts to establish communities to review findings and make sure that they're reproducible.

Thank you. So, this is an issue. It was brought to the fore by claims, probably justified claims, by certain pharmaceutical companies that many of the observations on which they might have relied for the development of drugs were, when published, were irreproducible. Amgen claimed that, I don't know, 70 or 80% of the papers that they thought they could rely on for compounds that were promising turned out not to have reproducible results that sort of completely eliminated the utility of the observation. So, unfortunately, Amgen had no reason to pursue this and to publish their observations. That was not part of their business plan. They didn't want to have to go to the trouble of publishing things. To justify their conclusions. And so unfortunately, this has given rise to a lot of concern, justifiable concern, that the biomedical enterprise may be resting on shaky ground. So one effort that we're involved with at eLife is called the Reproducibility Project. It's funded by a private outfit that got a charitable donation and that has identified— this particular group has identified the 50 most important or most highly cited papers in cancer biology in the past few years. And they have commissioned laboratories to replicate key experiments in each of these papers. And they approached various journals asking for a partner, a publication partner, to present the replication studies in two stages. An initial stage is what are called a registered report. It's a report written by the group that has been convened to replicate the experiments to define exactly which experiments and exactly how they will conduct the research. And these have been submitted, many of them now have been submitted and reviewed. Most of them have been accepted. Many have been published in eLife. And the groups that have been approved are now going forward to actually do the experiments. Then when they're done, they'll come back with their findings, which will be once again reviewed, peer reviewed, by our editorial board and by ad hoc referees. So, we will systematically explore the key experiments in many, but probably not all, of these papers. This is an enormous expense and— expensive and lengthy enterprise, but I think it's a beginning to see whether the claim made by Amgen and others really can be justified. Now, another component of your suggestion is, should— many of us feel that journals that publish results that prove to be irreproducible should be willing to accept publications that challenge those results. And unfortunately, particularly I have to say with the luxury journals, they are very reluctant to do so. I'll give you an example. Science published now 5 years ago a paper, an outrageous paper, that claimed that phosphate in the backbone of DNA phosphate was replaced by arsenate in a brine microorganism growing in a salt flat in California. This paper had been reviewed by geologists but not by microbiologists. And any microbiologist who looked at the paper realized that the authors had taken no particular pains to remove phosphate from the water that they used to grow this organism. And so it was almost certainly true that low level of phosphorus had been scavenged and— so it was dismissed. And there were many critical comments made, but Science never retracted that paper. 5 years later, they have never retracted that paper. I think journals bear some responsibility to correct the mistakes that they make. And our position at eLife is if we publish a paper that is erroneous, we will publish a suitably a justified challenge to that paper in the form of a short report. And we've done so already for a couple of papers and will continue to do so. And I think all journals should bear that responsibility. So we have to be part of the self-correcting mechanism to prove to the public and to our funding agencies that we can manage science. Otherwise, it will be managed for us. Yes? [INAUDIBLE] more perceived importance. Would ELife itself also fall as part of this, where even a scientific contribution that's not significant enough might get turned down? So how do you try to— Well, it's a delicate balance. You raise, of course, an important point. And I'm often asked, what's to keep ELife from becoming yet another luxury journal? Well, there are two things— there are several things that I mentioned and I'll highlight again. First of all, I think we're not in the business of selling magazines. Our business plan is we lose money. We spend the money that our donors give us. So we're not trying to hype, you know, by selling copy. Secondly, all decisions, all editorial decisions are made by active scientists. Not by professional editors. So when a paper is initially reviewed, it's reviewed by an expert in that field. It goes first to a senior editor and that senior editor will then confer with one or more members of a very large board of reviewing editors covering all of biology or with outside experts to determine whether there's something new and significant and whether it's been done competently in the paper. So we are selective. We have to triage 60% to two-thirds of the submissions that come in that contain observations that our scientists feel are not significant, but rather incremental. So we don't think we're going to be subject to that, and we will not have any artificial limits on the number of papers that we accept or the number of pages in a paper that we accept. We don't have a target number of 10% or fewer. We're happy to accept as many really good papers as we receive. [Speaker:STUDENT] But how do you define significant enough content? [Speaker:PROFESSOR] Yeah, well, that is a subjective judgment. There's— we, you know, I don't believe in impact factor, so it's a subjective judgment. And the thing that we've done is to assemble a group of editors who have the same attitude about this and who are experts in their field. And who make those determinations. They make those kinds of— we all make those kinds of subjective determinations. And the trouble is, of course, if we were, you know, to copy +1 and just accept everything that was considered accurate, people would not send their best work to us. We feel that there needs to be an alternative venue for important work that does not follow the commercial model of Cell and Nature to some extent science, but rather is done by active scholars. And that is what distinguishes us from those other journals. And— but we're going to have to be selective to—

in order to attract the best papers. Yes. Thank you again for your talk. What role do you think academic institutions play in setting priorities promotions and tenure policies. It seems to me that young scientists are still, despite having these alternative journals now to publish in, are still caught in some very traditional outdated policies that force them to seek prestige or seeming prestige if they do.

Well, of course, and it's often put to me rather bluntly than you have, you know, a young Scholars will say, "I'm a victim and, you know, this is a system that you created and I can only respond because I have no choice." Well, your question is quite simple. Who's responsible? It is the leaders of this institution, the department chairs, the committee chairs, the medical school dean. All of those leaders set the standards by which you as a scholar are evaluated. And I— wherever I go now, I call on them to develop more reliable criteria to evaluate your scholarly output. Let me give you some good examples of where this is going at a quite high level. The NIH now has changed the statements that investigators produce to describe their past work. It's no longer just a list of papers. You have to identify key key papers that you published in the recent grant period, and then to describe succinctly exactly what makes these papers important. Likewise, for many years, the Howard Hughes Institute, when investigators are reviewed for renewal, they ask their investigators to identify the 5 most important papers during the preceding 5 years, and then these papers have to be described in a paragraph that could be understood by a broad audience of reviewers. And I can tell you from experience that, in fact, that's exactly what the review team looks at. They don't look at the number of papers, the impact factor, I guarantee you, is not considered. They read the descriptions of the 5 papers that an investigator puts forward, and then they go into the papers in detail, and they probe those details in questions. In an interview after— when the investigator is up for renewal. I think that can be done at all stages. When you are considered for fellowship or for promotion, you can produce a document that describes succinctly exactly what your key contributions are in the form of a narrative that even an administrator could understand. So I think that's what we should do. I think frankly we should move away from these silly numbers. There are many other ridiculous numbers. There's this H-index, which is supposed to be a measure of one's cumulative contributions, but it's greatly influenced simply by one's age and number of publications and not necessarily the content. So I don't think it can be measured by a single number, but rather by a narrative. Indeed, I would suggest that we each take responsibility to craft a narrative, a brief concise narrative of our most important contributions that could be evaluated at each stage in our career. I believe, for instance, that when a job is posted for a faculty position and there may be hundreds of applications for that position, a broadly knowledgeable committee could look through every application and read what I would call an impact statement that the candidate would put forward that says, "This is me. This is what I've done. This is the important work that I've done." And on the basis of that alone, I believe a committee could actually rank order candidates to select a short list for closer inspection of the papers, of letters of recommendation, and the like. Instead, what happens now is people take the lazy way out. They use as a surrogate, "Wow, he's got 3 Nature papers," and they haven't bothered to read the paper at all. That is a completely inadequate measure of scholarship, and I call on anyone here in such responsible positions to evaluate exactly how they do business. Hi. Hold it down. Can you hear me? Yep.

Thank you for your wonderful talk. I'm just starting my scientific career, and I'm already cynical because I keep hearing about how the peer review system is broken and how the journals make you pay to submit your work and then pay to read your own work. And if you have any words of encouragement for me and other young scientists, I'd be greatly honored.

Well, you know, It's not a bad life. I mean, I'm a pretty happy person. It wasn't always so easy. My first NIH grant was completely trashed because I had no preliminary results, I had no experience with yeast, I was not a geneticist. But, you know, one muddles along. The problem is that you may be reflecting everything that I've been saying for the last 20 minutes, and that is this pressure. To get your work published in one of these few venues. That's really, that's really a source of the problem. And now these journals, they're very good business people, they've created a kind of a cascade of journals. And so if you don't make it into Nature, well, we'll just transfer your reviews over to our second-ranked journal and/or our third-ranked journal. And oh, by the way, it costs more to publish in those journals. And so they've captured, they've captured you. And I think this is— I think this— that is the broken aspect of the system, which, you know, I'm trying to do something about. [Speaker] I'd just like to hear some of the personal side of, you know, when you got the phone call, did you still use telephones or did people email or text you or whatever and, you know, travel the rigmarole. Okay, well, there is a rigmarole. So, I had just returned from Germany the Sunday night before what I now refer to as the blessed event occurred. And so, I was tired and I went to sleep at 9 o'clock, but not before my wife and her caregiver— my wife is quite ill— had found an article that was online from Stockholm in the AP written by a reporter who wrote an article entitled "Five Discoveries That Have Not Yet Won the Nobel Prize." And Rothman and I and Higgs— Peter Higgs was another one— were on that list of five. I don't remember who the other three were. And I said, "It's irrelevant. It's gossip. It's meaningless. Go to bed." So I fell asleep, fortunately. And then at 1:20 in the morning, you know, when you're— I suppose if you were in Hawaii, it would be even worse, maybe it would be better, but at 1:20 in the morning, I got a call from Corinne Hansen, who is the secretary of the Karolinska Committee. But, you know, when you get a call at 1:20 in the morning, it's usually bad news, right? But— Well, for better or worse, when you've sort of, you know, been nominated and people tell you and people warn you You know when— what day it is. Indeed, I have to say, you know what hour it's going to be. So— but I was sleeping. So— but my wife heard the phone first and she blurted out, "This is it!" So I stumbled out of bed and I went to the phone and there was this very reassuring Swedish accent on the other side. And I knew it wasn't a hoax because I actually recognized the voice. I actually worked with Hanson in another committee. And he congratulated me, and in great eloquence, the first words out of my mouth were, "Oh my God." Hear from an a-religious person. Anyway, so there were a few questions, a few comments, and then Hanson said, "Oh, and don't tell anybody." What do you mean don't tell anybody? I said, "Well, what about my family?" Oh, okay, you can tell your family, but there's gonna be a press conference in an hour in Stockholm, and so they can't tell anybody. So I hugged my wife and got on the phone, and I called my 87-year-old father. That was a moving experience. My mother died some years ago, and increasingly in recent years, my father, as October arrives, reminds me that he's not going to live forever and this better happen. But that particular year he'd lost track of the day and so when I called him, his new wife shrieked and answered the phone and he was awakened. So that was moving and I called my children and my best friends and I called Jim Rothman. We compared notes and then a few minutes later Adam called and we had a little interview about what was going to happen and, you know, my reaction. And then it was rather peaceful for the next 45 minutes after I'd made those calls until at the moment of now 2:30 in the morning, which was when the press conference was held in Stockholm, you know, basically the stuff hit the fan. I had been warned years earlier by our press officer at Berkeley to call him at home if this happened. So I had his phone on my— number on my cell phone, so I called, awakened him. And by 3 o'clock, I had 2 press officers from Berkeley in my home lining up the TV camera crews out in the front of the house. I had my landline in one ear and the cell phone in the other ear. It was bedlam and has remained so for the last roughly 2 years. So there was a press conference at Berkeley that day where I was presented with my parking permit that you heard about and a call from the president of the University of California system. And that evening our closest friends wanted to take us out to dinner and Our friend Patty suggested she could get a reservation at Chez Panisse. I don't know if you know this restaurant, but it's a very fancy restaurant. It's really one of the great restaurants in the area. And it was inconceivable that she could get a reservation so late on that day. So she called and they said, "Are you kidding? There are no rooms." And she said, "Well, what if I bring today's Nobel laureate? Would you have space?" "Oh, well, in that case, we have a table." So, okay, life has been like that since then. So there's more, but there are better things to discuss.