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A Prize for Membrane Magic

Suzanne R. Pfeffer1,* 1Department of , School of , Stanford, CA 94305-5307, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2013.11.014

The 2013 in or Medicine has been awarded to , , and Thomas Su¨ dhof ‘‘for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells’’. I present a personal view of the membrane trafficking field, highlighting the contributions of these three Nobel laureates in a historical context.

Our story begins in 1974, when I was a UC transport and localization in the cell.’’ transport from the endoplasmic reticulum Berkeley undergraduate. Jim Rothman But how proteins were transported from (ER) to the Golgi (Baker et al., 1988) and in was studying the properties of phospho- their site of synthesis to the cell surface 1990, published a careful double-mutant lipids in membrane bilayers with Eugene was not yet known. electron microscopy study that ordered Kennedy at Harvard, and Randy Schek- In 1976, Schekman began his lab as all the SEC gene products. Because man, who had just completed a PhD at an Assistant Professor at the University vesicles accumulated as intermediates Stanford University for work with Arthur of California at Berkeley, and chose to upon loss of the function of certain SEC Kornberg on DNA replication, joined the study protein secretion in baker’s yeast. gene products, this study was the first lab of Jonathan Singer at UC San Diego I first met Schekman then, as a biochem- to demonstrate the role of discrete to study protein mobility in cell mem- istry major, working in a lab on the same transport vesicles as true intermediates branes. Singer and Garth Nicholson had floor. Lee Hartwell, then at the University in the process by which proteins move just published a fluid mosaic model for of Washington, had just reported his use through the secretory pathway in yeast. the organization of lipids in bilayers. It is of yeast to identify the genes Schekman and his colleagues spent hard to imagine that the simple structure responsible for driving the cell-division the next several years cloning the genes of cellular membranes was still being cycle, which yielded Hartwell a Nobel encoding SEC proteins and examining debated at that time. During the same Prize in Medicine or Physiology in 2001 their functions in driving vesicle transport year, George Palade, with with and . Today, from the ER to the Golgi complex. His and , was awarded the yeast is a very popular experimental work led to the discovery of the COP-II Nobel Prize in Physiology or Medicine system, but in 1976, it was not at all clear coat that drives this process (Figure 1C) for his groundbreaking electron micro- that yeast would contain a secretory (Barlowe et al., 1994). The fundamental scopy studies of protein secretion from pathway or whether secretion in yeast importance of this discovery is best the exocrine . It was Palade would in any way, reflect pathways used appreciated when one considers that who established the concept that proteins by human cells. Schekman and a grad- more than one-third of the human synthesized on membrane-bound ribo- uate student, Peter Novick, took a very genome encodes proteins that must somes are transported, vectorially, into bold step and established a set of traverse the secretory pathway, and the lumen of the endoplasmic reticulum conditional mutant yeast strains that COP-II-coated vesicles carry them from (ER) before transport to the Golgi com- were temperature sensitive for cell-sur- the ER to the Golgi. Nothing was known plex and secretory storage granules for face growth (Novick and Schekman about the molecular basis for this process subsequent export from cells. In 1974, 1979). These strains were termed, sec before Schekman’s pioneering work. And the concept of the secretory pathway mutants for secretion mutants. the Schekman lab environment was so being used to create the limiting mem- Subsequent work by Schekman and encouraging of ‘‘collaborations’’ that a brane of cells was still only a supposition. colleagues identified 23 complementation number of lab member pairs got married And although Palade surmised that the groups and electron microscopy con- during this period. abundant, small vesicles that surrounded firmed that cells bearing sec mutations Two years after Schekman arrived at the Golgi complex in his electron micro- accumulated vesicles or other UC Berkeley, James Rothman started graphs participated in transport between when grown at the nonpermissive tem- his lab at Stanford University. Also membrane compartments, this was not perature (Figure 1A) (Novick et al., 1980). inspired by and his yet fully established (Palade, 1975). Later, inspired by his earlier training with colleagues in the Department of Gu¨ nter Blobel solved the first step of Arthur Kornberg and the success of Biochemistry, Rothman took a biochem- the Palade pathway, and he received the James Rothman and colleagues in recon- ical approach and set up a cell-free Nobel Prize in Medicine or Physiology in stituting membrane traffic events (see system to study ER-to-Golgi transport 1999 ‘‘for the discovery that proteins below), Schekman and coworkers estab- using mammalian cell components (Fries have intrinsic signals that govern their lished a cell-free system to study protein and Rothman 1980). Subsequent work

Cell 155, December 5, 2013 ª2013 Elsevier Inc. 1203 Figure 1. Milestones from the Path to the Prize (A) Accumulation of secretory vesicles in mutant cells at the permissive (top) or nonpermissive temperature (bottom) (from Novick et al., 1980). (B) Purified COP-I vesicles that mediate intra-Golgi transport and transport from the Golgi to the ER (Malhotra, V., et al. [1989]. Cell 58, 329–336). (C) Purified COP-II vesicles that carry proteins from the ER to the Golgi (Barlowe et al., 1994). (D) A synapse from embryonic cultured hippocampal neurons showing normal synaptic vesicle morphology (Janz, R., et al. [1999]. Neuron 24, 1003–1016). A fraction of vesicles are docked at the active zone, poised for rapid release in a highly calcium dependent manner. revealed that the reconstituted reaction he was seeking. We now know that fractions and test their activity, it is likely represented transfer of a glycoprotein so-called COP-II-coated vesicles, discov- that none of the fractions will be active from one Golgi stack to another. Three ered by Schekman, are responsible for because the other nine essential compo- papers spearheaded by Bill Balch and this process, in all , large and nents are no longer there. Every assay Bill Dunphy in the Rothman laboratory in small. And Vivek Malhotra and Rothman needed to be carried out using biochem- the mid-1980s reported a more stream- were the first to show that COP-I vesicles ical complementation—and lucky for lined assay and showed that the donor mediate protein transport within the Golgi Rothman, only one component was and acceptor membranes for the reaction stack (Figure 1B). These independent sensitive to NEM. This meant that assays represented distinct stacks of Golgi discoveries in the Schekman and Roth- of different fractions could be carried out membranes (cf. Balch et al., 1984). I man laboratories would not have been in the presence of a small amount of worked in Rothman’s lab as a postdoc in possible without the help of the electron NEM-treated cytosol to reveal the desired 1984 and 1985 when this work had just microscopist, Lelio Orci. activity. been published; I followed the subse- A very important breakthrough came The next breakthrough came when quent developments closely as a new when Rothman and coworkers purified Rothman and colleagues cloned the faculty member in a nearby lab. the first that was needed for gene encoding the NSF protein and found Many cell biologists were very skeptical the in vitro transport reaction they had it was homologous to the SEC18 gene of the in vitro findings that Rothman reconstituted (Block et al., 1988). They product discovered by Schekman and reported in the mid-1980s. The work identified a protein that had an N-ethyl- coworkers to be essential for membrane represented the first reconstitution of a maleimide (NEM)-sensitive, active site transport in yeast (Wilson et al., 1989) membrane trafficking step in the test thiol group that was needed for the and had been cloned and sequenced tube, and up to this time, microscopists in vitro transport reaction. The protein earlier by Scott Emr, a former Schekman rather than had dominated was named, NSF for NEM-sensitive fac- postdoc. This discovery demonstrated the field. Complicating matters was the tor. While today this sounds straightfor- that the process that Rothman had recon- fact that Rothman is a forceful visionary ward, imagine that you have an assay for stituted reflected a physiological and who was so eager to move the field for- a process that requires addition of crude highly conserved process. Now, two ward that a few mistakes were made cytosolic proteins, and ten proteins are entirely independent lines of investigation along the way. For example, at one point, provided by this crude cytosol. If you could proceed synergistically to reveal Rothman believed that clathrin-coated want to purify those proteins, you might the molecular events mediating these vesicles carry proteins from the ER to use chromatography to separate cytosol fundamental cellular processes. Doug the Golgi. A few missteps did not deter according to charge or size. But as soon Clary in Rothman’s group next identified Rothman from later finding the answers as you separate the cytosol into different a set of soluble proteins that are needed

1204 Cell 155, December 5, 2013 ª2013 Elsevier Inc. for NSF binding to membranes (soluble 25 (discovered by Michael Wilson and Chemie in Go¨ ttingen, Germany. He then NSF adaptor proteins or ‘‘SNAPS’’) (Clary not yet known to be needed for exocy- worked as a postdoctoral fellow with et al., 1990). These turned out to be tosis). Rothman’s use of brains as a Mike Brown and Joe Goldstein at the homologous to the yeast SEC17 gene source of abundant membranes was a University of Texas Southwestern Medi- product. This finding was, again, very lucky choice because rat brain synaptic cal Center studying the LDL receptor important—Schekman’s work provided components were already well studied gene—its cloning and regulation. In 1986 the genetic proof for the significance of by neuroscientists. he joined the faculty at the University the Rothman biochemistry. Mark Bennett and Scheller had just of Texas Southwestern Medical Center shown that the presynaptic plasma mem- and returned to his neuroscience roots, Discovery of SNARE Proteins—the brane protein, syntaxin binds to another applying newly learned molecular Fusion Machinery synaptic vesicle protein, synaptotagmin. skills to this important field. With Reinhard In October 1992, Giampietro Schiavo and Rothman proposed that NSF helped drive Jahn, Su¨ dhof cloned a number of synap- Cesare Montecucco published a paper membrane fusion by acting on a complex tic vesicle proteins, and he showed that showing that the target of tetanus and of a vesicle protein (in this case VAMP1) Rab3 GTPase was a synaptic vesicle botulinum-B proteolytic neurotoxins was bound to a target protein, syntaxin. The protein; he also discovered the protein, the synaptic vesicle protein, VAMP1/ So¨ llner/Rothman experiment represented complexin, which is a critical regulator of synaptobrevin. This demonstrated that the first functional possibility that these SNARE pairing, and the protein Munc18 VAMP1, a protein first discovered by proteins represented the players actually that is an essential component of the from electric fish and driving membrane fusion. synaptic vesicle fusion protein complex. later found in mammals as synaptobrevin In collaboration with Richard Scheller, Munc18 is an SM (Sec1/Munc18-like) by Tom Su¨ dhof and Reinhard Jahn, was Rothman went on to show that the protein, a family of proteins that control essential for synaptic vesicle release. Soluble NSF adaptor protein receptors the availability and ability of SNARE But this finding did not provide a mecha- (VAMP, SNAP25 and syntaxin, named proteins to form fusogenic complexes nism by which VAMP1 participates in SNAREs) formed a tight complex that for all membrane traffic events (Su¨ dhof synaptic vesicle release. In early 1993, could be dissociated by NSF protein and Rothman, 2009). So¨ llner and Rothman (So¨ llner et al., (So¨ llner et al., 1993b). More information, Since the discovery of synaptotagmin 1993a) published a landmark paper that including contributions from Bill Wickner, in 1991, a major focus of Su¨ dhof’s work provided the key clue as to how transport made it seem more likely that NSF’s role has focused on understanding how this vesicles could fuse with specific targets. would be to help recycle SNARE proteins protein provides calcium regulation to Rothman and coworkers had shown after a fusion reaction. Rothman was the process of synaptic vesicle release. that membrane transport in vitro required later able to fully reconstitute a fusion Synaptotagmin contains two protein 2+ the membrane association of NSF, medi- reaction using only purified SNARE pro- kinase C-like C2 domains that bind Ca . ated by the soluble NSF adaptor proteins teins (Weber et al., 1998). The subsequent Su¨ dhof showed that synaptotagmin or SNAPs. They knew that NSF was an three-dimensional structure of a SNARE binds to both phospholipids and SNARE ATPase and that ATP hydrolysis released complex by Axel Brunger and colleagues complexes in a Ca2+-regulated manner. NSF from membranes in vitro. They thus in 1998 cemented the notion that forma- Although synaptotagmin is not abso- searched for the membrane associated tion of complex between SNARE proteins lutely required for synaptic vesicle exocy- ‘‘receptor’’ for the SNAPs and NSF, on vesicles and on target membranes tosis, Su¨ dhof and coworkers showed that reasoning that the receptor would be a was the very likely mechanism by which synaptotagmin is required for rapid and membrane component needed for vesicle membranes fuse. And several of the yeast coordinated synaptic vesicle release. In fusion. Rothman’s colleagues incubated proteins needed for secretion were ho- very elegant experiments, he went on to immobilized, recombinant NSF with puri- mologous to the synaptic vesicle SNARE prove that synaptotagmin functions as fied SNAP protein, together with a deter- proteins, providing a homologous set of the key calcium sensor by showing that gent extract of rat brain membranes proteins to drive all of the specific mem- mice expressing mutant synaptotagmin as a source of the NSF/SNAP receptor. brane fusion events that take place in proteins with altered calcium affinity They first incubated the mixture under the cell cytoplasm. displayed altered calcium sensitivity in conditions in which ATP could not be neurotransmitter release (Ferna´ ndez- hydrolyzed (ATPgS), and then added Calcium Regulation in Dallas Chaco´ n et al., 2001; Su¨ dhof and Roth- Mg-ATP and collected the ATP-released Tom Su¨ dhof enters this story somewhat man, 2009). It is the ability of the process material. The remarkable finding was later, and although he is now my Stanford to respond to calcium that enables neu- that a discrete set of proteins eluted colleague, most of the work for which rons to secrete neurotransmitter rapidly from the column—and some were already he is honored was carried out before he and precisely (Figure 1D). known to be synaptic vesicle constituents arrived in California. Su¨ dhof received the Over several decades, Schekman and (VAMP1/synaptobrevin), a component Dr. Med. degree in 1982, studying the Rothman provided the first identification of the presynaptic plasma membrane, secretory granules of neuroendocrine of the molecular machinery needed for syntaxin (cloned earlier by Akagawa cells and a calcium-binding protein from transport of proteins through the secre- as well as Richard Scheller), and a electric rays, with Victor Whittaker at the tory pathway, a process they showed is synaptosome-associated protein, SNAP- Max-Planck-Institut fu¨ r Biophysikalische conserved from yeast to humans. They

Cell 155, December 5, 2013 ª2013 Elsevier Inc. 1205 each chose a risky path and embraced important ways to the discoveries sum- zola, M., Amherdt, M., and Schekman, R. (1994). a research question of fundamental marized here. But I believe that I speak Cell 77, 895–907. importance where they could contribute for the entire field of membrane trafficking Block, M.R., Glick, B.S., Wilcox, C.A., Wieland, essential molecular detail using a combi- when I say that this award was well F.T., and Rothman, J.E. (1988). Proc. Natl. Acad. Sci. USA 85, 7852–7856. nation of genetics and biochemistry. deserved. This month, Jim, Randy, and Working in parallel, they demonstrated Tom have reminded all of us of the impor- Clary, D.O., Griff, I.C., and Rothman, J.E. (1990). Cell 61, 709–721. the importance of transport vesicle inter- tance of membranes and membrane Ferna´ ndez-Chaco´ n, R., Ko¨ nigstorfer, A., Gerber, mediates in the secretory pathway and trafficking in all of medicine and physi- S.H., Garcı´a, J., Matos, M.F., Stevens, C.F., Brose, the mechanisms by which such vesicles ology. And just as Palade left us with N., Rizo, J., Rosenmund, C., and Su¨ dhof, T.C. form from the ER (Schekman) and Golgi many unanswered questions, there is (2001). 410, 41–49. (Rothman). Their work revealed the roles still much to be discovered about how Fries, E., and Rothman, J.E. (1980). Proc. Natl. of so-called SNARE proteins in vesicle proteins move through the secretory Acad. Sci. USA 77, 3870–3874. fusion with target membranes for all pathway, how the pathway is established, Novick, P., and Schekman, R. (1979). Proc. Natl. compartments of the secretory and and how the pathway is regulated in Acad. Sci. USA 76, 1858–1862. endocytic pathways. As for the molecular health and disease. Congratulations to Novick, P., Field, C., and Schekman, R. (1980). Cell basis of synaptic vesicle release, Tom these pioneers! 21, 205–215. Su¨ dhof made pivotal contributions, along Palade, G. (1975). Science 189, 347–358. with others with whom he has previously ACKNOWLEDGMENTS So¨ llner, T., Whiteheart, S.W., Brunner, M., Erdju- been honored. Su¨ dhof and Richard ment-Bromage, H., Geromanos, S., Tempst, P., Scheller were corecipients of the 2013 Research in the Pfeffer lab is supported by the NIH, and Rothman, J.E. (1993a). Nature 362, 318–324. the American Association, and the Ara and they also shared the So¨ llner, T., Bennett, M.K., Whiteheart, S.W., Parseghian Medical Research Foundation. 2010 Kavli Award for Neuroscience with Scheller, R.H., and Rothman, J.E. (1993b). Cell 75, 409–418. Rothman. These men were the first to REFERENCES Su¨ dhof, T.C., and Rothman, J.E. (2009). Science clone and characterize the proteins 323, 474–477. present in purified synaptic vesicles, Baker, D., Hicke, L., Rexach, M., Schleyer, M., and Weber, T., Zemelman, B.V., McNew, J.A., Wester- obtained and characterized by several 54 Schekman, R. (1988). Cell , 335–344. mann, B., Gmachl, M., Parlati, F., So¨ llner, T.H., and labs, including Reg Kelly’s at UCSF, with Balch, W.E., Dunphy, W.G., Braell, W.A., and Rothman, J.E. (1998). Cell 92, 759–772. 39 whom I worked as a PhD student. Rothman, J.E. (1984). Cell , 405–416. Wilson, D.W., Wilcox, C.A., Flynn, G.C., Chen, E., No single award can recognize all Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Kuang, W.J., Henzel, W.J., Block, M.R., Ullrich, the individuals who contributed in very Hamamoto, S., Salama, N., Rexach, M.F., Ravaz- A., and Rothman, J.E. (1989). Nature 339, 355–359.

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