Since most molecules are too large to directly pass through membranes, a mechanism is required to deliver the cargo

The 2013 Nobel Prize in Physiology or Medicine is awarded to Dr. James E. Rothman, Dr. Randy W. Schekman and Dr. Thomas C. Südhof for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells. This represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalisation, organises the routing of molecules packaged in vesicles to various intracellular destinations, as well as to the outside of the cell.

Specificity in the delivery of molecular cargo is essential for cell function and survival. This specificity is required for the release of neurotransmitters into the presynaptic region of a nerve cell to transmit a signal to a neighbouring nerve cell. Likewise, specificity is required for the export of hormones such as insulin to the cell surface.

While vesicles within the cell were long known to be critical components of this transportation scheme, the precise mechanism by which these vesicles found their correct destination and how they fused with organelles or the plasma membrane to deliver the cargo remained mysterious. The work of the three 2013 Laureates radically altered our understanding of this aspect of cell physiology.

Eukaryotic cells differ from prokaryotic cells by their more complex intracellular organisation. Distinct cellular processes are compartmentalised. This improves efficiency but a problem emerges. Different compartments need to exchange specific molecules and certain molecules need to be exported to the cell exterior. Since most molecules are too large to directly pass through membranes, a mechanism is required to deliver the cargo.

Randy W. Schekman used yeast genetics to dissect the mechanism involved in membrane and vesicle trafficking. He used baker’s yeast (Saccharomyces cerevisiae) as it secretes glycoproteins. Also, the genetically amenable organism was ideal to study vesicle transport and fusion. Schekman devised a genetic screen to identify genes regulating intracellular transport.

Initially, he identified two genes — sec1 and sec2 — and then went to further identify 23 genes that belonged to three different classes. The sequence of posttranslational events in the export of yeast glycoproteins was then determined with the aid of mutants. By studying the genetic and morphologic study of these mutants, Schekman discovered vesicle intermediates in the traffic between the endoplasmic reticulum (ER) and Golgi apparatus. Importantly, the sec17 and sec18 mutants accumulated small vesicles implicating a role in vesicle fusion.

James E. Rothman embarked upon a novel approach to dissect events involved in intracellular vesicle transport using an in vitro reconstitution assay. By using this approach, he purified essential components of the vesicle fusion process. Since it was difficult to express genes in animal cells in the 1970s, Rothman took advantage of a system based on vesicular stomatitis virus (VSV).

In this system, large amounts of a particular viral protein, the VSV-G protein, are produced in infected cells. A unique feature of this system is that the VSV-G protein is marked by a particular sugar modification when it reaches the Golgi compartment, which makes it possible to identify when it reaches its destination.

He then studied both vesicle budding and fusion, and purified proteins from the cytoplasm that were required for transport. The first protein to be purified was the N-ethylmaleimide-sensitive factor (NSF). The next important protein was SNAP (soluble NSF-attachment protein). SNAPs bind to membranes and assist in the recruitment of NSF. An important point of convergence between Schekman’s and Rothman’s work was the discovery that one of the yeast mutants, sec18, corresponded to NSF, thus revealing that the vesicle fusion machinery was evolutionarily ancient.

Using the NSF and SNAP proteins as bait, Rothman next turned to brain tissue, from which he purified proteins that he later named SNAREs (soluble NSF-attachment protein receptors). The three SNARE proteins — VAMP/Synaptobrevin, SNAP-25 and syntaxin — functioned together in the vesicle and target membranes. These SNARE proteins were discovered earlier but their functions were unknown.

VAMP/Synaptobrevin was found on the vesicle, and SNAP-25 and syntaxin were found at the plasma membrane. This prompted Rothman to propose the SNARE hypothesis. According to the hypothesis, the different SNAREs found on the vesicles (v-SNARE) and the targets (t-SHARE) played a critical role in vesicle fusion — through a set of sequential steps of synaptic docking, activation and fusion.

Aside from testing his hypothesis in vitro, he provided evidence that the system has a high degree of specificity, such that a particular target SNARE only interacted with one or a few of the large number of potential vesicle –SNAREs.

In essence, Rothman dissected the mechanism for vesicle transport and membrane fusion, and through biochemical studies proposed a model to explain how vesicle fusion occurs with the required specificity.

Thomas C. Südhof who was a junior group leader at the University of Texas Southwestern Medical School in Dallas set out to study how synaptic vesicle fusion was controlled. While Rothman and Schekman provided the fundamental machinery for vesicle fusion, how the vesicle fusion was temporally controlled still remained enigmatic. This is important as vesicular fusions in the body need to be kept carefully in check, and in some cases vesicle fusion has to be executed with high precision in response to specific stimuli. This is the case for example for neurotransmitter release in the brain and for insulin secretion from the endocrine pancreas.

Südhof elucidated how calcium regulates neurotransmitter release in neurons and discovered that complexin and synaptotagmin are two critical proteins in calcium-mediated vesicle fusion.

Complexin competes with alpha-SNAP, but not synaptotagmin, for SNAP receptor binding. Neurons from complexin knock-out mice showed dramatically reduced transmitter release efficiency due to decreased calcium sensitivity of the synaptic secretion process. This revealed that complexin acts at a late step in synaptic fusion as a clamping mechanism that prevents constitutive fusion and allows regulated exocytosis to occur.

Südhof also discovered synaptotagmin-1 (21), which coupled calcium to neurotransmitter release. The role for synaptotagmin-1 as a calcium sensor for rapid synaptic fusion was established by elegantly demonstrating that calcium binding to synaptotagmin-1 participates in triggering neurotransmitter release at the synapse. He also characterized Munc18-1, which corresponds to Schekman´s sec-1 and is therefore also called an SM protein.

SM proteins are now known to be an integral part of the vesicle fusion protein complex, along with the SNARE proteins. Südhof showed that deletion of Munc18-1 in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles.

In effect, Südhof made critical discoveries that advanced the understanding of how vesicle fusion is temporally controlled and he elucidated the ways that calcium levels regulate neurotransmitter release at the synapse.

Importance for medicine

The work of Rothman, Schekman and Südhof has unravelled machinery that is essential for routing of cargo in cells in organisms as distantly related as yeast and man. These discoveries have had a major impact on our understanding of how molecules are correctly sorted to precise locations in cell. In the light of this, it comes as no surprise that defects at any number of steps in the machinery controlling vesicle transport and fusion are associated with disease.

Vesicle transport and fusion are essential for physiological processes ranging from control of nerve cell communication in the brain to immunological responses and hormone section.

Deregulation of the transport system is associated with disease in these areas. 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. Furthermore, immune cells in our bodies rely on functional vesicle trafficking and fusion to send out substances including cytokines and immunologic effector molecules that mediate.

(Edited excerpts from the scientific background available at nobelprize.org)

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