Randy Schekman - Berkeley - Part 1 - Studying protein secretion in yeast

Hello. My name is Randy Schekman. I am in the department of Molecular and Cell Biology at the University of California at Berkeley. What I'd like to tell you about today is a fascinating area of modern cell biology where we study how the cell surface grows and how intracellular organelles are constructed. This process is assembled by a fascinating pathway called the secretory pathway. This process is used by all cells from bacteria to man to deliver proteins molecules and lipid molecules to different destinations. within the cell. I hope to persuade you today that this process can be studied using classic techniques, genetics and biochemistry, to reveal a mechanism that is fundamentally conserved in all organisms, particularly in those that have a nucleus, so called eukaryotic organisms. Now to begin with I'd like to focus on an organ that we are all very familiar with and love dearly, and that is our brain. The brain communicates using a pathway linked to protein secretion that involves the transmission of chemical molecules, so called neurotransmitters between adjacent nerve cells. And though it seems remarkable to say this, I hope to persuade you that the detailed molecular mechanism that allows chemical neurotransmitters to pass from one cell to another employs the same process that lowly yeast cells use to enlarge their cell surface. But to set the stage for this, let me take a look in greater detail within the brain to illustrate the basic unit of communication: the synapse where neurotransmitter chemicals are allowed to flow from one cell to another. This is a section cut through a nerve cell that captures the basic unit that I would like to describe. This is a nerve cell that has been chemically fixed and then embedded in a plastic resin that allows a diamond knife to cut a clean slice straight through, after which membranes may be highlighted with a chemical dye. So this is a nerve cell and it is connected in this instance to an adjacent cell, probably a muscle cell, through a narrow gap, the synapse, a clear area between adjacent plasma membranes of the two adjoining cells. Now focus if you will on these little packets. These are the basic unit of transmission in the brain and of protein secretion in all cells. It is called a vesicle and it consists roughly of two parts, a membrane bilayer, very much like the bilayer around the surface of the cell, and a clear interior content that contains in this case chemicals that are going to be secreted out of the cell into this gap, into the synapse. In the resting state, cells produce these vesicles and deliver them to a particular site on the plasma membrane where the vesicles come very close, almost touching the plasma membrane, but the membrane has not yet merged with the plasma membrane. These vesicles then are considered to be docked on the plasma membrane. They are then available for stimulation, and at the right moment, as you will see in the next slide, the vesicle membrane joins hands with the plasma membrane of the cell by a very important process called membrane fusion. At which point the interior content of the vesicle is delivered topologically to the outside of the cell, such that the content of the vesicle is secreted outside of the cell. Now this is more apparent in an image shown in rapid sequence of events on stimulation of the nerve cell, as you will see in this slide. So here in a very favorable example again this nerve cell just about ready to communicate with its neighbor produces a vesicle fusion event where the membrane of the vesicle has now merged with the plasma membrane. The two bilayers, lipid bilayers, are now continuous. and the internal content of this vesicle, the chemical neurotransmitters that are going to be secreted, are now in physical contact with the outside of the cell. A moment later the membrane appears to become almost continuous with the plasma membrane of the cell. But also in favorable examples this membrane must be recycled and so the content, although it has been secreted, consists of the membrane part that is reused. So these membranes can be taken back into the cell re-supplied with chemical neurotransmitters in the cytoplasm of the cell bind back to the cell surface to dock awaiting a new stimulation to produce yet another membrane fusion event. Now we can look at this in a very different way using a different kind of electron microscopic technique that allows one to visualize the surface on the outside of the cell by freezing the cell very rapidly and then hitting it with a hammer to crack the bilayer. It is possible to observe surface structure right at the moment of membrane fusion. Here we see two images shown looking down onto a nerve cell. In an area on the other side of the membrane if you were able to look inside of the cell, you would see vesicles, packets of neurotransmitter, awaiting instructions for membrane fusion, aligned very near particles that consist of membrane proteins embedded in the nerve cell plasma membrane. So now we are looking down into the nerve cell. In the resting state the rest of the membrane looks very smooth. Now it's then possible to stimulate the cell to achieve membrane fusion and synaptic discharge. And one sees in a very rapid sequence of events discharges that are observed as holes or dimples in the membrane. So if you picture in your mind's eye looking onto the surface of the nerve cell, looking down at the moment of stimulation, when the cell wants to transmit its neurotransmitter, you can see these dimples consisting of the interior content of the vesicle now spilling neurotransmitters outside of the cell, flowing away from the cell like this. Now there are literally thousands of investigators around the world who have for well over a hundred years studied this process in ever greater detail, with great sophistication, using the electron microscope and the tools of electrophysiology. However, until about 25 years ago it became, it was not possible to study this process at the level of molecules. To understand how the membrane actually achieves its binding to the plasma membrane, how a vesicle becomes docked, and how the membranes merge. And I'd like to tell you about two strategies that have been developed in a number of laboratories to study this process. And I'll focus on the work that has been going on in my laboratory for the last 30 years where we have studied the mechanism of vesicle production, vesicle movement, and vesicle fusion using genetics in a very simple organism, baker's yeast, Saccharomyces cerevisiae. Now let's have a look at the baker's yeast cell to get a handle on what this cell is capable of. Of course, it is not a brain, it doesn't secrete neurotransmitters. But as you'll see, yeast cells growing in the wild on the surface of a grape, or growing in the laboratory, must use the very same process that I have described to transmit newly synthesized molecules, both proteins and phospholipids, to their site on the surface of the cell. So here we see a cluster of yeast cells. This might be in fact a population of cells that have just been scraped off the surface of a grape. You can see they are slightly oval cells. They are all very homogeneous. They can be grown in the laboratory in very large quantities, studied as individual single cells or studied as pure populations of cells. There are traditional techniques of genetics that I will describe that can be used to study any process in this cell. Techniques that are very much simpler as applied to yeast than to human cells. Now yeast cells grow and divide a little bit different than an animal cell. You see here for instance an example of a yeast cell that has produced a bud. Yeast cells grow by a process of budding. The mother cell, after it has reached a certain size, and it has evaluated the environment and decided to commit itself to another cell division event, begins to elaborate a bud on its surface, and this bud enlarges for the next hour, hour and a half, until it becomes approximately the size of the mother portion of the cell. And you can see in this populations, this population, different examples of budding yeast cells caught at different stages in the process of bud enlargement. Finally after an hour and a half or so, when the bud is approximately the same size as the mother cell, it separates by a process of fission, producing a daughter cell and the remaining mother cell. Once again if the nutrient conditions are satisfactory, both mother and daughter can elaborate new buds, and thus a population can continue to grow exponentially as long as the nutrients are there and available for constructing macromolecules, replicating chromosomes, making lipids, and so on. Now one gets a better impression of the process that is used to assemble this bud surface by cutting a section through the yeast cell just as we saw when we cut a section through a nerve cell. Here for instance is an example of a yeast cell, in this case the experimentalist has very conveniently provided labels that identify the membranes in this otherwise now shadow of a cell. This cell has been chemically fixed, embedded in plastic, and sectioned. Here on the top is the bud. This was the surface structure that I showed you in the previous slide. That grows, this would be fairly early in a new cell division event where the bud is a little smaller than the mother portion of the cell. On the very outside of the yeast cell there is a rigid cell wall that consists of polysaccharides, chitin molecules that are found in plants, and that distinguish a yeast cell from an animal cell. This provides the yeast cell with its rigidity and allows it to survive in the wild. Now within the cell, inside the cell one sees membranes that are very similar to those found in animal cells. For instance, yeast cells have a nucleus. They are a bona fide eukaryote. The nucleus has a membrane envelope consisting of two membranes and you'll see this in another example in a moment. The yeast cell also has a digestive organelle, called a vacuole. This organelle is similar to an organelle called a lysosome in animal cells. And yeast cells use this organelle to digest macromolecules that it wants to get rid of and recycle components like sugars and amino acids. Now more to the point of our discussion there are several additional organelles that one can see in a normal, rapidly dividing yeast cell that are characteristic of the secretory process. For instance, we see a strand of membrane emanating from the nuclear envelope, projecting into the cytoplasm. A thread of membrane to an envelope that is called the endoplasmic reticulum. This is a membrane involved in the biosynthesis of macromolecules that will end up leaving the cell. This organelle assembles polypeptides. The polypeptides pass from the cytoplasm, made by ribosomes in the cytoplasm, pass through the bilayer of the endoplasmic reticulum and then reside at least initially in this densely stained interior of the organelle. Yeast cells also have another structure characteristic of mammalian cells though in normal yeast cells it is not as obvious as in a mammalian cell. That is a structure called the Golgi apparatus, and I will point to that in a few minutes at... where it becomes more evident when traffic is interrupted at this station. The Golgi apparatus is a bus station. It receives material from the endoplasmic reticulum and sifts molecules according to their final destination, transferring some to the cell surface, others to the vacuole, and others simply cycling back and forth between the Golgi structure and the nuclear envelope or the endoplasmic reticulum. It's an elaborate, very interesting organelle that was discovered by classic cytologic techniques in the 19th century. And even simple yeast cells have this structure, a bus station en route to the cell surface. Finally in a rapidly growing cell there are small vesicles seen here because of the staining technique used for this image as little particles underneath the plasma membrane of the bud portion of the cell. And I hope to persuade you that these small vesicles are very similar to the vesicles that are responsible for neurotransmitter secretion. In this case, not secreting neurotransmitters, the vesicles are instead responsible for the discharge of proteins that become part of the cell envelope or the cell wall or membrane proteins that become integral to the plasma membrane of the cell. So one imagines that these vesicles, the result of an assembly line process of events, are delivered by a track into the bud where they dock and fuse and execute that last essential element, in this case of growth. In the nerve cell this process results in neurotransmitter secretion without net cell growth, but in the case of a yeast cell the logic is simply to produce these vesicles continuously to allow the envelope to grow in preparation for the bud maturing to become equivalent to a mother cell. This can be indicated in another example by evaluating the surface structure of the yeast cell just as we did the surface structure of a nerve cell. So here we take yeast cells and freeze them rapidly and hit them with a hammer to cut right through the bilayer on the surface of a bud. We can see, just as in the case of the nerve cell, dimples enriched on the surface of this bud this bulb growing out of the mother portion of the cell where the dimples represent the fusion events that are responsible for cell surface growth, much as you saw a few moments ago dimples on the surface of a nerve cell permitting neurotransmitter secretion. Now a simple cartoon will illustrate the principle that I would like to use to understand how this process works. Here we have a normal yeast cell shown on the left with vesicles containing cargo molecules indicated by little dots and a membrane, a bilayer surrounding these particles. These packets, these vesicles are delivered to the bud portion of the cell where occasionally a vesicle will find its right target and the membranes will merge and the process of fusion permits the membrane of the vesicle to become part of the plasma membrane of the cell. With time, as these vesicles are delivered, the cell enlarges until the mother portion of the cell and the bud portion of the cell are equivalent. Now this very simple, perhaps almost trivial, cartoon illustrates an essential point, and that is if one were to interrupt the flow of vesicles, their production, their targeting, their docking, their fusion at the plasma membrane, if somehow one were able to interfere with that process, one would expect vesicles to build up inside the cell at the expense of cell surface expansion. So many years ago a very talented graduate student by the name of Peter Novick joined my lab at UC Berkeley with this very goal in mind. To try to interfere with this process using a traditional form of genetics. Let me tell you how one can study a process that should be essential for cell viability. Now of course as I have drawn this example, if one were to interfere with this process by deleting an essential gene involved in conveying vesicles to the bud, you would expect the cell to die. So how can you study a dead cell? One classic approach that allows one to investigate an essential gene is to make mutations in that gene that interfere with its function at a high temperature, but not interfere with its function at a low temperature. These are so called conditional or temperature sensitive mutations, and one can understand this very simply. If you take a protein that is stable over a range of temperatures and introduce a mutation very often on a surface residue in the molecule, and if the mutation causes a substantial change in the amino acid in an essential part of the molecule, this sometimes creates a molecule that unfolds at higher temperature. That is it is thermally unstable. And if that protein molecule is essential for a cell process, then of course the cell cannot survive exposure to the high temperature where this protein unfolds. It turns out, as you will see in a moment, the process of protein secretion indeed depends on such genes. And it was possible to define these genes by exposing yeast cells to chemical mutagens that introduce random mutations into individual genes throughout the yeast genome, and then using a variety of techniques to identify the mutations that specifically affect secretion. So one can arrest this process by taking yeast cells, which grow at a range of temperatures on the surface of the grape, and they may grow as low as ten degrees centigrade. In the laboratory one very often will grow yeast cells at room temperature or at body temperature, 37 degrees, and that useful range of temperature can readily distinguish normal yeast cells from cells that harbor a thermo sensitive mutation in an essential gene. Well, after some effort Peter Novick was able to define a gene, called sec-1. Temperature sensitive mutations in this gene produce a molecule that is thermally unstable and confer on a sec-1 mutant cell the ability to grow and secrete at room temperature, but not at body temperature, at 37 degrees. And you'll see the dramatic effect of this mutation in the next slide. So you might recall several slides ago a normal yeast cell with a smattering of organelles throughout the cytoplasm and a very small cluster of vesicles in the bud portion of the cell. In this case, sec-1 temperature sensitive mutant cells have been incubated at body temperature, 37 degrees, for several hours. Under conditions where the wild type cells would have grown, and divided, doubled, more than doubled, but in this case, the cell is arrested because vesicles, no longer restricted to the bud portion of the cell, now fill up the entire cytoplasmic volume. Thousands, many thousands of vesicles, a many fold higher concentration of vesicles than one sees in a normal yeast cell are arrested because of a single amino acid substitution in an essential residue in the sec-1 protein molecule. At higher magnification, again in the same cell, you can see that these vesicles are indeed membrane enclosed. There is a double track bilayer appearance that is readily apparent on some of these vesicles. And subsequent experiments showed that these vesicles when isolated from broken sec-1 mutant cells carry the set of protein molecules that would be delivered to the cell surface. They carry the membrane proteins, the sugar transporters, or they carry the molecules that become part of the cell wall. They carry them in the vesicle, but they can't merge with the cell surface because of a defect in the sec-1 protein molecule. Now another very useful feature of these mutants, one that is convenient for investigation is that fact that many of them, many of these mutations produce a molecule that though it may unfold at body temperature, is not so defective as to preclude refolding of the mutant protein when the cell is returned from body temperature to room temperature. So if you take a cell, such as this, warmed to 37 degrees centigrade, and then cool the cell back down to room temperature, the misfolded sec-1 protein molecule can refold to form its proper functional conformation and the vesicles that had accumulated at the high temperature now re-engage the cell surface and can achieve membrane fusion and discharge. Thus the mutant cells resume growth and the intracellular accumulation of traffic material is discharged and the cell can go along on its merry way. However, of course, if the cells are kept at the high temperature for a very long period of time, they die of a kind of molecular constipation. They cannot continue to grow and they choke. Now using this property, that is the accumulation of material within the cell at the expense of enlargement of the cell surface. Peter Novick realized that it may be possible to isolate a lot more mutants to define many more genes in this pathway relying on the property of these cells becoming dense. That is the buoyant density of the cell increases substantially during this incubation at 37 degrees. The density of the cell increases so much so that the cells, mutant cells that block this pathway can be separated from normal, wild type yeast cells on a density gradient. So a density gradient technique was used to produce and isolate many more sec mutant cells defining over a couple of dozen new genes. In evaluating these mutants by techniques such as electron microscopy, it became apparent, after about a year or so, that there were at least ten different genes encoding ten different protein molecules, and we now know many more than that, that are required at the very same stage in this process as sec-1. That is where the protein molecules cooperate to permit the vesicle to dock and fuse with the plasma membrane. Over a period of many years these ten genes were cloned, the wild type gene was closed, and the corresponding molecules were identified in mammalian cells, and a very nice connection between this process in yeast and the process in mammalian cells, indeed in nerve cells, was established by comparing the sequence of the yeast protein to that of the mammalian protein. And just for purpose of this comparison, I have illustrated a set, a subset, of the molecules required at this last step of the pathway in yeast, and the corresponding step where synaptic vesicles in a nerve cell dock and fuse with the plasma membrane of the nerve cell. Let me highlight just a few of these molecules. Sec-1 shown here in this circular orange shape is, as I have shown you in the last slide, essential at that last step, we know a great deal about how this molecule works, what it touches to initiate the process of membrane fusion. And we know that equivalent molecules, identified here as munc18 in the nerve cell or nSec-1 according to other investigators, serves an absolutely conserved role in this process of joining membranes, vesicle and plasma membranes, to initiate the fusion event. In both instances, and in many other locations in the cell, this sec-1 molecule engages two membrane proteins, integral membrane proteins, one in the vesicle membrane and one in the plasma membrane, which form the junction that allows the membranes to become so closely apposed, the bilayers to touch so closely, that membrane fusion occurs then very rapidly. So this is a fundamentally conserved process, first revealed by genetics in yeast, and then subsequently by a great deal of molecular cloning analysis and biochemical analysis in mammalian cells. Now, the sec-1 characteristic of vesicle accumulation was seen in many mutants, but not in all of them, and I'd like to show you two other examples of mutations that affect other stations in the secretory pathway. One, a mutant called sec-7, had a very surprising and quite distinct effect on the intracellular organization of membranes in cells incubated at 37 degrees centigrade. You'll recall from some slides ago that a normal cell has thin tubules characteristic of a Golgi apparatus, but in this mutant, sec-7, the structure blows up to become an elaborate network of tubules stacked one on top of another almost like a stack of pancakes. This is unusual. It is essentially never seen in a wild type cell. and the interpretation of this, shown here in higher magnification is that some essential function is served by the sec-7 protein molecule to permit proteins to move out of the Golgi apparatus into a secretory vesicle. In fact, it is possible using a simple genetic test to demonstrate, to document, that assertion. If one takes a yeast cell that has a mutation that produces this characteristic and introduces into that cell another mutation, the sec-1 mutation, that by itself would cause vesicles to accumulate, and then the double mutant cell is shifted from room temperature to 37 degrees, the structure that accumulates in such a double mutant cell is this organelle, rather than the vesicles that you saw in sec-1. The interpretation of that double mutant analysis is that this station precedes the vesicle station. That is this station must execute its function before vesicles can be produced. Indeed when these cells, sec-7 mutant cells are returned from 37 degrees, down to room temperature, this structure essentially dissolves and gives rise to vesicles that then are targeted to the cell surface. Finally, one last phenotype seen in a number of genes, initially nine and now almost thirty different genes, produces an exaggerated endoplasmic reticulum. You'll recall from the thin section of a wild type yeast cell, thin electron dense tubules using a different staining procedure than this instance, we see that these tubules are very much more elaborate. They have enlarged the lumen, the clear interior is now much wider than normal. Correspondingly, the envelope of the nuclear membrane becomes much more readily apparent. And these tubules wind around through the cytoplasm making a much more extensive network than is apparent in a wild type cell. And you can see in this blow up of that image that this structure comes very close to, almost touching, the plasma membrane. Here again one can show that the organelle, the endoplasmic reticulum, becomes engorged with molecules that must pass to the next station along the secretory pathway. And if one takes this mutant, and combines it with the sec-7 mutation, that double mutant accumulates this structure, rather than the Golgi structure, which suggests that this structure precedes in its function the Golgi station. So another very large number of genes required to process material at this earlier stage in the secretory pathway. Well finally, let's put this all together in the form of a simple cartoon that illustrates the contour of the secretory pathway in yeast. and to illustrate this similarity with the same process in mammalian cells. At the very beginning, in the lower left hand corner of the diagram, you see a ribosome inserting a protein into the membrane of the nuclear envelope, or the endoplasmic reticulum, a set of genes discovered by another graduate student, Ray Deshaies, defines the machinery in the endoplasmic reticulum membrane responsible for acquiring molecules that will initiate the secretory event. Later on, proteins are sorted, as you'll see in the next chapter of this presentation, into vesicles that bud from the membrane of the endoplasmic reticulum and deliver content to this next station, the Golgi apparatus, indeed as you'll see later, there is a bidirectional flow of material back and forth between these two stations. Within this bus station, the Golgi apparatus, molecules are sifted, some are diverted by an intracellular route to the vacuole, the yeast equivalent of the lysosome, by a very elaborate machinery most elegantly described by Scott Emr and Tom Stevens in their studies on the sorting event that achieves vacuole assembly. Other molecules, not diverted into the vacuole, instead become packaged into vesicles the kinds of vesicles that we saw accumulate in sec-1, and they are then delivered to the plasma membrane under the set of sec genes that define this last stage. in the secretory pathway. Now in the next chapter I will describe a rather more precise technique that allows one to focus with great precision on the mechanism of protein transfer. This pathway illustrated by genes and mutants highlighted the essential proteins, but it by itself says very little about what those protein molecules do. And in order to understand how membranes form, and how they fuse, we must go in with, in a way, a higher power microscope, using more precise tools and I'll tell you about that in the next chapter.