Nobel Prize: Physiology, Medicine

Cell transport system

Print edition : November 29, 2013

James Rothman.

Randy Schekman.

Thomas Südhof.

Fig. 1. Each cell in the body has a complex organisation where specific cellular functions are separated into different compartments called organelles. Molecules produced in the cell are packaged in vesicles and transported with special and temporal precision to correct locations within and outside the cell.

Fig. 2. Schekman discovered genes encoding proteins that are key regulators of vesicle traffic. Comparing normal yeast cells with genetically mutated ones in which vesicle traffic was disturbed, he identified genes that control transport to different compartments and to the cell surface.

Three American scientists, who elucidated the mechanisms involved in the transport of proteins and other essential biomolecules within the cell and through the cell wall to other target cells, win this year’s Nobel in Physiology or Medicine.

THE cell is the functional basic unit or the building block of life of all known living organisms. Cells contain many biomolecules such as proteins and nucleic acids, which are enclosed in a plasma membrane. There are about 100 trillion cells, on average, in the human body. The typical size of a cell is about 10 micrometres. While the largest is about 135 micrometres, the smallest is about 4 micrometres.

Bodily functions and survival thus depend on how effectively and efficiently cells can carry out their functions, which are to synthesise life’s essential proteins and other biomolecules, such as hormones, neurotransmitters, cytokines and enzymes, and deliver them to different destinations in the body for corresponding specialised functions. This year’s Nobel Prize in Physiology or Medicine has gone to two American and one German-American scientists who elucidated the mechanisms involved in the transport of proteins and other essential biomolecules within the cell and through the cell wall to other target cells. James E. Rothman, 63, of Yale University, Randy W. Schekman, 65, of the University of California, Berkeley, and Thomas C. Südhof, 58, of Stanford University, according to the Nobel citation, will share the prestigious award “for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells”.

Cells are classified as eukaryotic and prokaryotic. Eukaryotes contain a nucleus and prokaryotes do not. Eukaryotic cells are about 15 times wider in size and as much as 1,000 times greater in volume than a typical prokaryotic cell. While prokaryotic cells are usually single-celled organisms, eukaryotic cells can be either single-celled organisms or part of multicellular organisms. The major difference between prokaryotic and eukaryotic cells is the latter’s more complex intracellular structure. Eukaryotic cells contain membrane-bound compartments, which include the nucleus itself and other organelles, in which specific metabolic activities take place. This compartmentalisation is known to vastly improve the efficiency of many cellular functions and prevent potentially dangerous molecules from moving freely within the cell and affecting cellular functions. However, since most biomolecules are too large to break the membrane barrier directly, the exact mechanism by which organelles exchanged molecules and the cell exported specific ones with requisite specificity in their transportation and delivery needed to be understood.

For example, specificity is essential for the release of neurotransmitters into the presynaptic region of a nerve cell to transmit the signal to the neighbouring cell. Similarly, specificity is required for the transport of hormones such as insulin to the cell surface for it to be released into blood.

The use of the phrase “machinery” in the Nobel citation for the cellular transport mechanism is interesting. In his post-award interview to Nobel Media, Rothman said: “That is exactly how I think about it, machinery. And the reason is that one of the major lessons in all of biochemistry, cell biology and molecular medicine is that when proteins operate at the subcellular level they behave in a certain way, as if they were mechanical machinery. It’s absolutely fascinating. When we study chemistry, the rules of chemistry, electrons, and so on, they operate at an even smaller level of atoms and molecules. But when you get to the sort of level of the nanoscale, you find that these objects start behaving as if they were mechanical. Exactly how I think about it, and always have…. My orientation originally was in physics. I was trained as a physicist as a young man. And what so attracted me about molecular biology is the opportunity to find the simplicity through that very simple concept.…”

It was long known, essentially because of George Palade’s work, that the vehicles involved in this cellular transportation machinery are tiny sac-like objects enclosed by a lipid bilayer, called vesicles, in which this molecular cargo is packaged and dispatched (Figure 1). It was the advent of electron microscopy and the associated new straining techniques that enabled visualisation of membranes within human cells, and in Schekman’s words, “it was [Palade’s] genius to realise, [and] to appreciate, how proteins that are going to be exported from cells are assembled in a kind of assembly line process inside the cell”.

Routing of molecules

The spatial and temporal precision with which the vesicles are delivered to the correct locations within and outside the cell and how they fused with organelles or the plasma membrane, however, remained mysterious. How do these miniature bubble-like structures carrying large molecules know where and when to deliver their cargo? The work of this year’s Nobel awardees marks a paradigm shift in the understanding of how the eukaryotic cell, with its complex compartmentalisation, organises the routing of molecules packaged in vesicles to various intra- and intercellular destinations in the body.

The three laureates unravelled distinct and critical components of the cell transportation machinery. While Schekman discovered a set of genes that were responsible for vesicle traffic, Rothman unravelled the protein machinery that enables the vesicles to fuse with their targets to permit the transfer of the cargo, and Südhof revealed how signals instruct vesicles to offload their cargo with precision. Together, Rothman, Schekman and Südhof have thus revealed the extraordinarily precise control system for the transport and delivery of cellular cargo. It has also been found that disturbances in this system can have deleterious effects and contribute to conditions such as neurological diseases, diabetes and immunological disorders.

Schekman was a PhD student of the Nobel laureate Arthur Kornberg, considered one of the greatest biochemists of the 20th century. He was apparently fascinated by how the cell organised its transport system. In the 1970s, he embarked on the study of its genetic basis, rather than its biochemical basis, by using yeast as a model system. Schekman realised that baker’s yeast ( Saccharomyces cereveisiae), which secretes glycoproteins, could be an ideal organism with which to study vesicle transport and fusion. He devised a genetic screen and identified yeast cells with a defective transport machinery resulting in vesicles piling up in some parts of the cell. He found that this congestion in the shuttle system was genetic and identified the mutated genes. He identified 23 genes, which could be assigned to three distinct classes depending on which parts of the cell they caused the traffic congestion in: endoplasmic reticulum or the Golgi complex or the cell surface (Figure 2). Essentially, these genes were thus responsible for three important facets of the cell transport system, and this discovery has provided new insights into the well-regulated machinery that mediates the vesicle transport.

Vesicle transport

Rothman, who was also intrigued by the nature of the cell’s transportation mechanism, chose the biochemical approach to determine the transport pathway. As a young group leader at Stanford in the 1970s, he took up the novel approach of studying the mechanism in vitro for which he developed a special assay to dissect the events involved in vesicle transport.

Rothman said in his post-award interview: “In the earlier years, when I started this project, at Stanford University, everybody told me it was nuts to go and try to reproduce the complexities, the mysterious complexities, that occur in a whole cell, in a cell free extract. And... my courage came from three sources, I would say. The first, in all seriousness, was youth. Because there’s certain arrogance in youth, I don’t [know] if I’d have had the courage to do that today. The second was the fact that, you could, in those days… do adventurous things with very little… preliminary data, and… still get support from the NIH [National Institutes of Health] to do it. And so in today’s environment I doubt very much I would have had the freedom or the opportunity to truly pursue this. And the third, frankly, was that I was inspired by… Arthur Kornberg.”

During his investigations on the problem of the cell’s transport system in mammalian cells in the 1980s and 1990s, Rothman discovered that a protein complex enabled vesicles to attach and fuse with their target membranes. The fusion process was analogous to the zipper action, with the proteins on the vesicles and those on the target membranes binding to each other like the two sides of a zipper. The specificity in terms of delivery to a precise destination was the result of the simple biological fact that there are many such proteins and they bind only in specific combinations. The same principle operates both within the cell and when the vesicle binds to the cell’s outer membrane to transport its contents to the exterior. It turned out that the proteins that Rothman identified in mammals corresponded to those encoded by some of the genes discovered by Schekman in yeast earlier. This is indicative of an ancient evolutionary basis for the vesicle transport system.

Schekman said in his Nobel media interview: “When I began my laboratory in 1976, really around the same time when Jim Rothman began his laboratory at Stanford, we conceived of very different approaches to try to identify the machinery responsible for this mechanism. And I used the technique of classical yeast genetics, to make mutations in yeast cells, that define the pathway, and Rothman used a very complementary biochemical process to reconstitute the pathway in vitro. Over a period of years... it became clear that he and I were working many of the same proteins, many of the same genes and proteins. So, that synthesis between the two sets really convinced us that we were on the right track, the project on, as I did.” Through his biochemical studies, Rothman was also able to propose a model to show how vesicle fusion occurred with required specificity.

Südhof was, on the other hand, interested in finding out how cells in the brain communicated with one another. He had originally been trained at the Georg-August-Universität and Max Planck Institute for Biophysical Sciences in Göttingen, Germany, and had come to work as a postdoctoral fellow at the University of Texas Southwestern Medical School in Dallas. Vesicles release neurotransmitters, the signalling molecules that they carry when they fuse with the outer membrane of the nerve cells according to the fundamental mechanism discovered by Schekman and Rothman. But these molecules are allowed to be released only under a stimulus from the nerve cells to its neighbours. A similar specific stimulus is required for endocrine pancreatic cells to release insulin into the bloodstream. In these cases timing is everything. Südhof was interested in finding out how this release was temporally controlled so precisely.

Calcium ions were known to be involved in this process, and in the 1990s, Südhof looked for calcium-sensitive proteins in nerve cells. He identified the molecular machinery that responded to an influx of calcium ions and triggered the vesicles to bind to the outer membrane of the nerve cell. Signalling molecules are released in accordance with the Schekman-Rothman mechanism. Südhof elucidated how calcium regulated the neurotransmitter release in neurons and discovered the two critical proteins involved in this calcium-mediated vesicle fusion. His discovery explained how precise timing of release of molecules was achieved in the cell’s transport mechanism and how vesicles’ contents were released on command. The essential machinery for routing molecular cargo within and outside cells that the trio have unravelled shows one important evolutionary aspect: that this mechanism is the same in organisms that are evolutionarily as different as enzymes and man. The specificity and precision involved in this vesicle transport and fusion mechanism also suggest that any defect in any of the steps in this complex machinery could lead to cells of different organs going out of sync, which, in turn, could lead to health disorders and diseases.

Deregulation of the vesicle transport system is now known to be associated with disorders in physiological processes ranging from control of nerve cell communication in the brain to immunological responses and hormone secretion. For example, metabolic disorders such as type 2 diabetes are characterised by defects in both insulin secretion from pancreatic beta-cells and insulin-mediated glucose transporter translocation in skeletal muscle and adipose tissue. Immune responses depend on vesicle trafficking and fusion to transport appropriate immunological biomolecules such as cytokines.

In addition, specific mutations in the genes encoding the proteins in the vesicle fusion machinery give rise to a number of diseases. For example, in certain forms of epilepsy, certain mutations in the gene encoding a protein called MUNC-18-1 (responsible for neurotransmitter secretion from synaptic vesicles) have been identified. Likewise, in a subset of patients suffering from familial hemophagocytic lymphohistiocytosis (FHL), mutations in certain other specific genes have been found. In FHL patients, natural killer cells (small cells that destroy virus-infected cells or tumour cells without activation by an immune cell or antibody) fail to regulate properly when encountering target cells, leading to hyperinflammation, which can be lethal. Furthermore, certain bacterial toxins target the vesicle fusion machinery.

Botulism, caused by the anaerobic bacterium Clostridium botulinum, is a paralytic disease, and the majority of the toxin types cleave to the essential protein complexes that Rothman identified and thus prevent the release of the key neurotransmitter molecule at the neuromuscular junction. Thus, the discoveries of Schekman, Rothman and Südhof have provided clarity to disease mechanisms and prospective treatments.