Session 4: Vesicle Trafficking

00:00:07.20 Hello, my name is Randy Schekman.
00:00:10.01 I'm at the University of California at Berkeley in the Department of Molecular and Cell Biology.
00:00:16.22 Today, I'll be giving three presentations on the cellular process that is used to
00:00:24.05 package protein molecules that are made inside the cell,
00:00:28.08 but have to be shipped outside of the cell.
00:00:31.09 In my first presentation, I'll discuss some of the historical aspects of how we learned
00:00:37.02 about biological membranes, and how they are deployed to encapsulate protein molecules
00:00:44.28 as they are made inside the cell.
00:00:47.19 In my next lecture, I'll describe how this process was understood at the molecular level
00:00:55.10 using a simple nucleated organism, a simple eukaryote called Saccharomyces cerevisiae
00:01:02.11 or baker's yeast, and I'll tell you about both genetic and biochemical approaches
00:01:08.12 to understanding this process.
00:01:10.24 And finally, in my last lecture, I'll discuss some very recent experiments on how cells
00:01:17.14 package small RNA molecules that are encapsulated into vesicles that are discharged at the cell exterior,
00:01:25.15 and may communicate between cells in our body.
00:01:30.23 But let's begin with a discussion of how cells are organized.
00:01:36.02 The basic principle of organization of cells comes with an understanding of the structure
00:01:43.27 of a biological membrane.
00:01:45.22 And that's depicted on my first slide.
00:01:48.22 So, here you see a cartoon of a biological membrane consisting of two leaflets of molecules
00:01:56.25 called lipids or phospholipids.
00:01:59.08 They are shown here with red balls that are the water-loving or hydrophilic head groups
00:02:08.00 of a phospholipid molecule, connected to these thin tails.
00:02:12.16 They are the water-hating or hydrophobic fatty acid side chains that constitute
00:02:19.21 the inner core of the membrane bilayer.
00:02:22.27 Two leaflets of lipids come together to form this bilayer.
00:02:28.15 Embedded in a biological membrane are these green structures.
00:02:33.02 They are protein.
00:02:34.12 They depict protein molecules.
00:02:37.02 Some of them go clear through the bilayer.
00:02:39.12 As you see here, this example is maybe, for instance, a channel in the membrane
00:02:45.15 through which small molecules may come and go.
00:02:48.12 Or this example may be of a protein that is a receptor, that sits on the outside of the cell
00:02:55.04 and recognizes hormones that may interact with a cell to convey information to the cell interior.
00:03:02.20 Now, all membranes in cells have this basic structure.
00:03:09.02 But each membrane in a cell has a different kind of personality.
00:03:13.20 And they go from very simple organizations to very complex organizations.
00:03:19.01 Here, for instance, is the simplest cell.
00:03:22.17 This is a red blood cell, coursing through your bloodstream.
00:03:26.27 It consists, at least in a human, of a single membrane surrounding an internal cytoplasm
00:03:34.18 that is filled with hemoglobin, the protein molecule in your red cells that carries oxygen
00:03:41.20 to your peripheral tissues.
00:03:43.09 So, just a simple cell with a single membrane.
00:03:47.20 But in contrast, for example, a much more complex cell.
00:03:52.26 This is a very important cell in your pancreas.
00:03:56.07 It's called the beta cell in the islets of Langerhans.
00:04:00.17 It's responsible for manufacturing insulin that is discharged outside of the cell,
00:04:09.15 carried within the cell by these little granules.
00:04:11.16 These... these look like little eye... eyespots, but they're granules that house insulin
00:04:19.13 and convey it through the cytoplasm to the cell surface, where it is discharged by a process
00:04:25.27 called membrane fusion, that we'll discuss in a few minutes.
00:04:30.06 So, enormous difference in complexity between a cell that has many functions, such as
00:04:36.21 in the pancreas, or a simple cell, such as the red blood cell.
00:04:40.14 Now, a cartoon of the various membrane organelles that are found inside of the cell is depicted here.
00:04:49.02 This is a cell from an epithelium surrounding a tissue that is responsible for making
00:04:56.01 many protein molecules that are shipped to different places, in and out of the cell.
00:05:02.26 At the base of the cell, you see the nucleus, housing the chromosomes.
00:05:08.19 Surrounding that nucleus are membranes that constitute a network called the endoplasmic reticulum,
00:05:15.24 that are dotted with these little particles.
00:05:19.03 They're ribosomes.
00:05:20.12 Ribosomes are the machines that stitch amino acids, one next to another, to make protein molecules
00:05:28.13 that are often transmitted across a membrane
00:05:33.04 into this clear space of the endoplasmic reticulum.
00:05:37.01 And we'll talk about that in a few minutes.
00:05:38.18 There are many other membrane organelles in the cell.
00:05:42.20 The powerhouse organelle, the mitochondrion.
00:05:45.13 A structure called the Golgi apparatus, through which protein molecules are conveyed.
00:05:51.12 And other membranes that have specialized functions, like the peroxisome or the endosome.
00:05:59.09 These all... all have biological membranes surrounding them and each has different protein molecules
00:06:06.23 that execute the unique functions of these organelles.
00:06:11.14 Now, much of what we know about the organization of an animal cell came from the pioneering work
00:06:18.26 of cell biologists in the middle part of the 20th century.
00:06:24.08 Prominent among them was a brilliant cell biologist by the name of George Palade.
00:06:29.11 Dr. Palade was an emigre from Romania.
00:06:34.02 He came to New York, where he established his laboratory at The Rockefeller University.
00:06:40.25 In the mid-1950s, it was Palade who discovered the ribosome.
00:06:46.15 He did this, as with much of the rest of the... of his work, by perfecting an instrument called
00:06:52.25 the electron microscope, which you see here... you see here him seated behind.
00:06:58.25 He and Keith Porter, and other scientists at the Rockefeller, devised procedures to
00:07:05.12 fix cells and tissues, and to preserve them so that they could be sectioned with a diamond knife,
00:07:13.09 and then visualized under an intense electron beam, where the electrons
00:07:19.16 were scattered by structures within the cell.
00:07:22.01 And all of the beautiful pictures, some of which I'll show you, were interpreted by him
00:07:27.12 to understand many of the functions of membranes that communicate with one another by the process
00:07:34.01 of protein secretion.
00:07:35.24 Now, let's go through, step by step, each of the organelles that Palade and his students
00:07:42.18 were able to appreciate, both by visualizing them in the electron microscope, but also
00:07:49.04 by isolating them and studying them as biochemical entities.
00:07:53.19 The first organelle that he was able to understand is the endoplasmic reticulum.
00:07:59.17 Here you see a section through a cell of the pancreas.
00:08:04.20 These cells in the pancreas are differentiated.
00:08:08.04 They are already developed to their full potential.
00:08:11.25 Their major role is in the production, packaging, and secretion of proteins that go in... eventually,
00:08:21.03 into the gut or into the bloodstream, and as a result the network responsible for
00:08:27.14 the manufacture of these proteins is highly elaborate.
00:08:30.25 In a cell of the pancreas that is differentiated to make proteins for export, the endoplasmic reticulum,
00:08:38.09 this network of membranes, can have a surface area that is 25-fold greater
00:08:45.14 than the surface area of the membrane that surrounds the cell.
00:08:49.02 So, it's an enormous and ela... and quite elaborate platform.
00:08:54.00 And you'll note that these platforms are studded with ribosomes, each of which is acting to
00:09:02.20 produce a protein molecule,
00:09:05.11 which will eventually find its way across the membrane of the endoplasmic reticulum
00:09:10.20 to rest in the clear luminal space.
00:09:14.05 This luminal space then represents a kind of a canal system within the cell, a large
00:09:22.27 fluid volume, collecting molecules that have passed the barrier of the endoplasmic reticulum membrane,
00:09:29.23 and are poised to be shipped along this canal network through the cell, by steps
00:09:36.10 that I will elaborate over the next few minutes.
00:09:38.28 Now, you can get a better sense of the dimensional arrangement of this endoplasmic reticulum
00:09:46.04 in the cartoon shown on my next slide.
00:09:48.00 You see here that it is not just a set of tubules, but it's actually a set of sheets
00:09:54.23 of leaflets that... envelopes that spread throughout the cytoplasm, and can occupy
00:10:01.18 a great fraction of the cell.
00:10:04.10 Most of this membrane has the ribosomes studding its surface, but there are also parts of it
00:10:09.23 that are smooth, that are free of ribosomes, that may represent transitional zones
00:10:16.17 from which molecules become packaged into vesicles that convey this material downstream in the pathway,
00:10:23.28 as you'll see.
00:10:26.06 Now, what Palade did to pursue this understanding of the function of the endoplasmic reticulum
00:10:34.02 was to devise techniques to break cells open, to take tissue, to homogenize, to break cells open,
00:10:41.04 and then to obtain partially and eventually highly purified fractions of membranes
00:10:48.06 that could be studied for their molecular composition and their biosynthetic potential.
00:10:53.25 Here is a very simple first step that Palade and his colleagues, Christian de Duve and
00:11:01.24 Albert Claude, devised to begin to fractionate membrane organelles.
00:11:06.06 So, one starts with a tissue, a pancreatic tissue, for instance, and this tissue
00:11:12.08 can be disrupted by a physical agitation to break the cells open, but to preserve membranes
00:11:18.20 relatively intact in a cell homogenate or cell lysate.
00:11:23.22 Now, in this lysate, if the cells have been gently broken, membranes retain
00:11:30.16 different sizes and shapes.
00:11:32.09 And they can begin to be separated from one another by a series of steps, of centrifugation steps,
00:11:38.18 where the homogenate is placed in a centrifuge tube and sedimented at different speeds.
00:11:44.24 At very low speed of sedimentation, large membranes, for example the nucleus,
00:11:51.16 sediment out of suspension to form a pellet at the bottom of the tube.
00:11:56.02 At medium speeds of centrifugation, other somewhat smaller organelles, like the mitochondrion,
00:12:01.24 the lysosome, or the peroxisome, can be sedimented and obtained in a slightly enriched form.
00:12:08.26 And then, at higher speeds of sedimentation, very small membranes, small vesicles,
00:12:15.15 eventually sediment out of suspension and form a pellet at the bottom of the tube.
00:12:19.24 And so these distinct pellet fractions can be examined for their biochemical composition
00:12:26.23 and for their structure, as seen in the microscope.
00:12:30.13 Now, another principle that Palade perfected to... specifically to isolate those membranes
00:12:37.16 that have ribosomes bound to them is shown on the next slide.
00:12:42.04 And this is a procedure where membranes are separated according not to their size
00:12:48.24 but to their buoyant density.
00:12:51.02 The membranes have distinctive buoyant density.
00:12:54.25 Membranes that are free of ribosomes tend to be more buoyant, less dense.
00:13:01.10 And they can be separated from membranes that retain ribosomes and... which are...
00:13:08.00 have a higher buoyant density.
00:13:09.19 So, in a homogenate, the sample having both membrane-bound and unbound structures
00:13:17.13 can be applied to the top of a gradient, typically a gradient of sucrose, from low to high.
00:13:24.28 And then the sample can be sedimented for a very long time so that the membranes
00:13:30.15 achieve an equilibrium buoyant density.
00:13:33.17 And the smooth membranes, lacking ribosomes, are... sediment to a position of low buoyancy,
00:13:41.13 whereas those membranes that have ribosomes sediment to a position of high buoyancy,
00:13:46.20 of high buoyant density.
00:13:48.22 Cleanly separating these two membranes.
00:13:51.08 This high buoyant density fraction is a relatively enriched source of membranes that have ribosomes,
00:13:59.14 and, as you'll see, have the ability to take protein molecules that are destined for secretion
00:14:08.01 and pass them across the membrane into the clear interior space of the organelle.
00:14:14.24 Now, I'm going to summarize work not only of Dr. Palade but principally of his protege,
00:14:24.04 another very famous cell biologist by the name of Gunter Blobel, who was able to
00:14:29.24 pursue Palade's original pioneering work using biochemical cell biology
00:14:35.26 to understand the precise mechanism
00:14:38.11 that proteins use as they pass from a ribosome across the membrane of the endoplasmic reticulum
00:14:45.14 into the clear interior space, the first step in a long sequence of events that eventually...
00:14:51.23 eventually will leave the protein molecules secreted outside of the cell.
00:14:56.05 So, here is, then, a summary of a great deal of work that Dr. Blobel achieved,
00:15:03.20 and for which he won the Nobel Prize.
00:15:07.21 We start with ribosomes that assemble together, a large subunit of the ribosome and a
00:15:13.28 small subunit of the ribosome.
00:15:15.18 They come together along with a messenger RNA, in this case, a messenger RNA that encodes
00:15:22.00 a protein that is going to be secreted.
00:15:26.22 What Dr. Blobel discovered is that proteins that are destined for secretion have a
00:15:34.22 special sequence at the very N-terminus, the beginning of the protein, that tends to be
00:15:40.04 somewhat apolar or hydrophobic.
00:15:43.12 And that sequence draws... called a signal peptide, draws the ribosome/messenger RNA/nascent
00:15:53.23 protein chain eventually to a channel in the ER membrane, through which the polypeptide
00:16:01.05 is inserted and progresses into the clear interior space, the luminal space of the ER.
00:16:08.24 In the course of the biosynthesis of this protein, Blobel discovered that the hydrophobic,
00:16:15.17 the apolar signal peptide, is clipped by a special protease in the ER membrane.
00:16:22.28 That produces the mature N-terminal domain of the secretory protein, that is now
00:16:29.16 free to fold into a functional tertiary structure in the lumen of the ER.
00:16:37.28 Folded properly and ready to progress through the pathway.
00:16:41.09 So, this call... eventually called the signal hypothesis, predicted the existence of a channel.
00:16:49.26 And in my next lecture, I'll tell you about how my laboratory was able to use genetics
00:16:56.01 to discover the genes that encode this channel.
00:17:00.23 Now, after molecules have folded and are ready to go, they are ready to perform their function,
00:17:10.19 eventually outside of the cell, they are recognized and conveyed in vesicles, that I'll describe
00:17:15.27 in my next lecture, to the next station in the secretory pathway, a structure called
00:17:22.11 the Golgi apparatus.
00:17:23.21 Here is a depiction of the Golgi apparatus.
00:17:26.03 It kind of looks like a stack of pancakes, although in three dimensions it's a rather
00:17:34.01 more complex organelle, where the membranes are interrelated, not only stacked
00:17:40.15 one on top of the other, but have tubular connections.
00:17:43.28 This was a structure that was first described in the 19th century by an Italian cytologist
00:17:48.26 by the name of Camillo Golgi, who...
00:17:51.26 whose discovery was based on his finding of a dye, a chemical dye, that highlighted
00:18:01.28 this membrane in nerve cells.
00:18:04.01 It highlighted this membrane at the expense of other membranes.
00:18:06.23 We now know that this dye that Golgi devised recognizes carbohydrate.
00:18:13.09 And carbohydrate is rich on glycoproteins that are packaged and conveyed through
00:18:19.00 the Golgi apparatus.
00:18:20.23 But, after this discovery in the late 19th century, very few investigators were able
00:18:28.04 to make progress.
00:18:29.17 For nearly 60 years, this organelle was considered a cellular curiosity with no obvious function.
00:18:36.24 And it was not until the 1960s and 70s, when Dr. Palade focused his vision on this structure,
00:18:43.22 were we able to deduce that it is a station, en route, between the endoplasmic reticulum
00:18:50.22 and the cell surface, through which molecules are conveyed.
00:18:54.06 Much as passengers would be conveyed through a bus station, they are conveyed through
00:18:58.25 the Golgi apparatus.
00:19:00.12 And shipped to different destinations in the cell and outside of the cell.
00:19:04.27 And I'll have more to say about this Golgi structure as time goes on.
00:19:09.24 Now, once molecules progress through this station, they are ready... they are mature,
00:19:17.06 they are ready to be encapsulated within granules that eventually convey them to the cell surface.
00:19:24.25 And there's a simple diagram that I'd like to share with you that describes what happens next,
00:19:29.27 after the Golgi apparatus.
00:19:31.14 So, here is a very simple depiction of the fate of secret... secreted molecules as they are
00:19:39.02 packaged into granules and eventually delivered to the cell surface.
00:19:43.04 So, here you see such a cartoon of a granule, that's got a membrane.
00:19:47.24 And the red dots on the in... on the inside represent molecules like insulin, that are
00:19:51.22 being manufactured inside of a beta cell of the pancreas.
00:19:56.00 At a certain time, this mature granule finds its way to the cell surface, and the membrane
00:20:02.25 of the granule merges with the membrane of the cell surface to form a continuous bilayer.
00:20:10.27 That results in the interior content of this granule being discharged to the cell exterior.
00:20:18.02 And crucially, this happens without breaking the cell, without breaching the permeability barrier
00:20:24.11 of the membrane that surrounds the cell, or else the cell would lyse.
00:20:28.15 So, you can then affect secretion of water-soluble molecules like insulin and hormones
00:20:36.27 and antibody molecules molecules by this process of membrane fusion.
00:20:42.14 And the final product, then, is seen outside of the cell.
00:20:46.12 Now, let's look at a real example from a cell that Palade visualized, showing virtually
00:20:51.24 the same thing that I've depicted in my cartoon.
00:20:54.26 At a certain crucial moment, the content of this granule, condensed in its interior
00:21:01.12 and surrounded by a biological membrane, migrates to the cell perimeter, where the two membranes,
00:21:08.13 the membrane of the granule and the membrane surrounding the cell, come to very close apposition,
00:21:16.06 so close that the cytoplasmic content between these two membranes is squeezed out.
00:21:22.13 The membranes come so close that they can approach each other within Angstroms.
00:21:27.10 And then, at a key moment, the cell receives a signal that causes the membranes to
00:21:34.05 merge by this process of membrane fusion.
00:21:36.27 And as you saw a moment ago, the interior of the granule is ejected to the cell exterior.
00:21:43.24 In this case, this granule is condensed and somewhat crystalline, but it dissolves
00:21:48.19 when it leaves the cell.
00:21:50.14 And eventually, protein molecules such as insulin are distributed into the bloodstream.
00:21:55.17 So, this is a crucial step that occurs not only in the pancreatic beta cells, but in
00:22:01.19 all cells, and virtually all cells that are manufacturing proteins.
00:22:05.17 Let me give you a couple of examples.
00:22:07.18 Here's a cell that contains a huge supply of proteins that are to be secreted.
00:22:14.04 Enormous numbers of granules are built up in this cell.
00:22:17.00 And eventually, when the cell is triggered by some stimulus... stimulant, to engage in
00:22:23.26 protein secretion, the granules all reach the cell perimeter.
00:22:27.27 And then look what happens, the cell almost appears as though it's exploding.
00:22:32.13 But the cell, in this case, still remains intact, but all of the material has been secreted
00:22:37.10 and the cell surface membrane is distorted by having accumulated a lot of this membrane
00:22:43.03 that was in granules, that now is at least temporarily merged and fused at the cell perimeter.
00:22:48.27 The cell restores itself, some of the excess membrane is taken back into the cell, it fills...
00:22:54.25 these granules are filled up, and the process can be repeated.
00:22:58.00 Now, in the brain, this process takes shape in the secretion of chemicals.
00:23:06.14 Not necessarily proteins, but chemicals, particularly chemical neurotransmitters.
00:23:11.08 And here's an example.
00:23:12.24 This is not a human brain, but this is actually the connection between a nerve cell and
00:23:19.01 a muscle cell at a structure called the neuromuscular junction.
00:23:22.22 This sample happens to be taken from a frog, but the same is true in all metazoan cells.
00:23:28.20 So, this is a nerve cell.
00:23:30.04 This is a nerve terminal.
00:23:32.15 The membrane that surrounds the nerve terminal is a... is a traditional plasma membrane.
00:23:38.00 But as you'll see, inside, in the cytoplasm of the nerve terminal, there are many small granules.
00:23:44.19 In this case, they're called vesicles or synaptic vesicles.
00:23:49.11 And these synaptic vesicles house the chemical transmitters that mediate communication
00:23:58.08 between a nerve cell and a muscle cell.
00:24:00.16 For instance, these synaptic vesicles house molecules like serotonin.
00:24:08.05 That affects mood and mood disorders in humans.
00:24:11.18 Or these synaptic vesicles may house dopamine, the chemical neurotransmitter that is responsible
00:24:19.27 for much of our movement and also affects cognition, and which is drastically reduced
00:24:28.21 in patients suffering from Parkinson's Disease.
00:24:32.13 Another very important neurotransmitter called acetylcholine, responsible for much of
00:24:38.09 the communication between nerve cells, and which is very tragically lost in patients that
00:24:46.28 succumb to Alzheimer's Disease.
00:24:48.17 So, these granules, then, are manufactured, they collect very high chemical concentrations
00:24:55.08 of neurotransmitters, and they come up right up to the cytoplasmic side of the membrane
00:25:01.26 surrounding the nerve terminal.
00:25:05.24 And you can actually visualize the process of fusion of these vesicles at the presynaptic membrane
00:25:13.12 by a very clever experiment that was first devised by John Heuser in St. Louis,
00:25:19.02 some years ago, that allowed him to stimulate a nerve terminal and then very quickly,
00:25:25.00 within milliseconds, capture images in frozen samples that allow one to actually see the membranes
00:25:33.08 begin to merge with the plasma membrane of the nerve cell.
00:25:37.15 Here is a time sequence.
00:25:39.21 A resting nerve cell, followed by stimulation and rapid processing.
00:25:45.11 Within five milliseconds, you can begin to see events where the vesicle has just
00:25:51.24 started to merge and the interior of the vesicle becomes secreted to the space, in this case,
00:25:58.22 between a nerve cell and a muscle cell.
00:26:00.16 The chemicals that diffuse into this cleft, the synapse, then bind on the muscle side
00:26:08.27 to receptors that allow a muscle cell, eventually, to contract.
00:26:13.04 So, all movement is based on this rapid communication of neurotransmitters, mediated by vesicles
00:26:23.02 that share much of the same process of secretion that we see in cells such as the beta cell
00:26:28.20 of the pancreas.
00:26:31.05 Now, Palade...
00:26:32.24 Dr. Palade didn't just take lots of pretty pictures, he did an amazing experiment
00:26:39.10 that allowed him to, in a way, visualize the stages in this process, step by step, using a combination
00:26:48.28 of pulse-chase radiolabeling, autoradiography, and thin-section electron microscopy
00:26:58.01 that gave us the picture that we now have, now 50 years later, of how this process is organized
00:27:05.04 in eukaryotic cells.
00:27:07.01 And this is, then, a simple cartoon that displays Palade's final pioneering work,
00:27:13.13 for which he won the Nobel Prize in 1974.
00:27:17.02 We know from his work that proteins originate on ribosomes bound to the endoplasmic reticulum.
00:27:23.14 They are allowed to fold in this clear interior space.
00:27:27.27 They are then packaged into little vesicles that convey material to the Golgi apparatus.
00:27:34.12 Material then flows through the Golgi apparatus.
00:27:38.23 Some is diverted from the Golgi apparatus to an intracellular organelle, such as the
00:27:44.21 lysosome, which is the... kind of digestive organ of a cell, where protein molecules
00:27:51.27 may be broken down.
00:27:53.02 Or, other granules are formed by budding at the Golgi apparatus to produce mature secretory granules
00:28:00.03 that move and, by a process of membrane fusion, discharge their content to the cell surface.
00:28:07.18 Now, I had the privilege of meeting Dr. Palade when I was a graduate student.
00:28:14.11 And then an important event in my career came when I was a postdoctoral fellow at UC San Diego.
00:28:21.26 And I heard Dr. Palade describe his pioneering work to an audience of
00:28:28.01 the American Society for Cell Biology.
00:28:30.28 This was in 1974, just as he had returned from Stockholm, having received his Nobel Prize.
00:28:37.26 And I was trained as a biochemist, not as a cell biologist.
00:28:41.28 It was clear how brilliant the work that Palade had done was.
00:28:47.02 And how revolutionary it was for the field of cell biology.
00:28:50.10 But, as a biochemist, what struck me was that this beautiful image, summarizing decades of work,
00:28:57.19 describing an obviously essential cellular process, was nonetheless devoid of
00:29:03.15 any molecular mechanistic understanding.
00:29:07.02 That is, in 1974, when Palade was recognized for his work, we didn't know about
00:29:16.08 any gene or protein molecule involved in organizing this pathway
00:29:20.11 -- nothing, literally nothing was known.
00:29:23.14 And so I resolved, when I began my career at the University of California at Berkeley,
00:29:27.16 to study this process in an organism that would allow a molecular dissection of
00:29:34.06 the mechanism of this pathway.
00:29:36.04 Everyone, until then, had studied mammalian cells or animals, where, at least in the mid-1970s,
00:29:45.16 the techniques of genetics and biochemistry were not well developed.
00:29:50.10 And so what I decided to do at the outset of my independent career was to explore this
00:29:55.13 process in a simple organism, baker's yeast.
00:30:00.02 Baker's yeast can be grown in large quantities.
00:30:02.01 Here is an image of a scanning EM picture of yeast cell, such as you might see growing
00:30:08.04 on the surface of a grape.
00:30:09.24 Yeast cells grow by a process of asymmetric budding, where a small bud emerges from the
00:30:18.08 surface of a mother cell and grows, in preference to the mother cell, during the first 90 minutes or so
00:30:25.17 of the growth of the cell, until the daughter cell approaches... achieves the size
00:30:33.23 of the mother cell, at which point they divide.
00:30:36.08 And if the nutritional conditions are correct, the cells can continue through yet another
00:30:41.07 cycle of division.
00:30:43.07 Now, yeast was a particularly important organism in the history of molecular biology because
00:30:50.03 of the use of traditional, classical genetic approaches that allow one to understand genes,
00:30:57.15 and thus proteins, that are involved in any cellular process, even essential cellular processes.
00:31:06.00 And I'd like to highlight, as a recognition of this application of genetics,
00:31:11.16 the work of a pioneering geneticist, a yeast geneticist by the name of Leland Hartwell, who was able,
00:31:19.05 using very simple visual techniques, to identify genes that are required for
00:31:27.05 the progression of cells through the cell division cycle.
00:31:30.11 He did this by introducing, using a chemical mutagen, mutations into the yeast genome.
00:31:37.10 And then looking among these mutants for those that affect a particular step in
00:31:43.11 the ability of the cell to complete its division cycle.
00:31:46.27 These are called cdc, or cell division cycle, mutants.
00:31:50.14 And each represents a mutation in a gene that is essential for cell viability.
00:31:57.02 The way you can study these genes is by obtaining mutations that are conditional in their effect,
00:32:02.05 that is, that allow the cell to grow at, for instance, room temperature, but kill the cell
00:32:07.27 when the cell is warmed to human body temperature, 37 degrees.
00:32:12.02 And so he obtained dozens of such mutations, each of which defines a gene required
00:32:18.05 for protein... for... for progression through the cell division cycle.
00:32:23.26 So, I'm going to conclude this first lecture with a simple image of a normal yeast cell
00:32:31.19 that gave my laboratory some inkling that yeast cells might be a good test system to
00:32:39.22 use the logic of Hartwell to discover the genes involved in protein export.
00:32:44.22 Here is a thin slice through a normal, a wild type, yeast cell, obviously very different
00:32:50.23 than a pancreatic cell.
00:32:53.19 It's dominated... the cytoplasm is dominated by a very high granular content of ribosomes.
00:33:00.02 But, as you can see, there are some organelles.
00:33:03.06 These are membrane organelles.
00:33:04.26 This large structure is the yeast vacuole.
00:33:08.00 It is the equivalent of a mammalian lysosome.
00:33:11.06 In other sections of this cell, you can see that the yeast cells have a nucleus.
00:33:15.21 You can also see that there are tubular membranes that are similar to the endoplasmic reticulum.
00:33:23.11 But my laboratory was particularly intrigued by the appearance of a cluster of small vesicles
00:33:32.07 that congregate under the bud portion of a dividing cell.
00:33:36.28 These vesicles seem likely to be responsible for conveying proteins for secretion into
00:33:43.07 the growing bud surface of the cell.
00:33:45.27 This is the bud of the cell.
00:33:47.25 But further, we imagined that the membrane of the vesicle would contain the building blocks
00:33:54.08 for the assembly of the plasma membrane.
00:33:57.13 And thus, by this process of membrane fusion, the vesicle would not only discharge proteins
00:34:04.28 into the cell wall that surrounds a yeast cell, but that the membrane of the vesicle
00:34:10.14 would be in a sense the building block of the plasma membrane.
00:34:14.17 So, the fundamental prediction that I'll leave you with in this part, and which I will elaborate on
00:34:19.12 in my next lecture, is that the genes involved in the production of these vesicles, we predicted,
00:34:26.28 would be required for cell growth and secretion.
00:34:31.12 And therefore the genes could only be studied by obtaining conditional or temperature-sensitive
00:34:37.10 lethal mutations.
00:34:38.10 So, we'll leave it there and pick it up in my next lecture.
00:34:41.10 Thank you.

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.