View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Leading Edge BenchMarks A Prize for Membrane Magic Suzanne R. Pfeffer1,* 1Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2013.11.014 The 2013 Nobel Prize in Physiology or Medicine has been awarded to James Rothman, Randy Schekman, 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 genetics 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 Albert Claude with Paul Nurse and Tim Hunt. Today, from the ER to the Golgi complex. His and Christian de Duve, 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 pancreas. 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 organelles 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 Arthur Kornberg 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 eukaryotes, 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 enzyme 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 biochemists 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.