In his first lecture, Dr. Randy Schekman overviews the secretory pathway and reviews historical experiments that shaped our molecular understanding of this pathway. The journey begins at the endoplasmic reticulum (ER), where proteins that engage the secretory pathway get translated. The mRNA of these proteins codes for a signal sequence that serves as a “tag” to bring the complex of the mRNA, ribosome, and newly synthesized protein to the ER for continued translation and movement of the new secretory protein across the ER membrane into the interior or lumen of the organelle. Vesicles transport the recently translated proteins to the Golgi Apparatus, where they get “packaged” and sent to their final destination.
In his second lecture, Schekman explains how his laboratory used baker’s yeast to uncover major proteins involved in the secretory pathway, and describes proteins involved in budding, vesicle trafficking, and vesicle fusion. Schekman also presents data from his laboratory that helped to identify the ER channel through which proteins enter the secretory pathway. These series of experiments show how, step by step, scientific knowledge evolves, uncovering the fundamental mechanisms to better understand human disease.
The Secretory Pathway: How Cells Package and Traffic Proteins for Export
Concepts: Secretory pathway, extracellular vesicles, endoplasmic reticulum, Golgi Apparatus, vesicle fusion, 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.
In this talk, Randy Schekman describes a genetic screen in yeast and then follow-up studies in vitro (in the test tube).
- Why would a scientist reconstruct vesicle budding in vitro when this phenomenon can be readily observed in live cells?
- What are some of the advantages of using biochemical techniques to study the secretory pathway?
Genes and Proteins Required for Secretion
Concepts: Secretory pathway, budding, vesicle trafficking, and vesicle fusion
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.
Professor, Molecular & Cellular Biology; HHMI Investigator
University of California, Berkeley Continue Reading
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