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Introduction

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Cells are structurally and functionally divided into a series of compartments among which the pathways of metabolism are precisely allocated, creating a network of specialized and complementary reaction vessels (see Protein trafficking). The nucleus, cytosol, mitochondria, endoplasmic reticulum, Golgi, plasma membrane, and lysosomes each carry out distinct biochemical pathways — for example, DNA synthesis, glycolysis, ATP synthesis, membrane biosynthesis, glycosylation of proteins, transport of solutes, and degradation of macromolecules, respectively — utilizing distinct sets of enzyme proteins. This view of the cell, established during the 1950s and 1960s, raised "the sorting problem," a defining issue for cell biology in the decades that followed: How does each compartment acquire and maintain its unique spectrum of proteins? The cell-free reconstitution of transport between compartments was pivotal in elucidating the molecular mechanisms involved.

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Background

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The sorting problem was ripe for attack by the late 1970s because of key findings that allowed it to be posed in reasonably precise terms. George Palade had discovered that protein transport can occur between otherwise separate membrane-bound compartments of the cell. Palade and colleagues showed that secreted (and probably other) proteins follow a transport route from the endoplasmic reticulum (ER) to the Golgi and then to the cell surface and suggested that membrane-bound vesicles mediate transport. Günter Blobel had just discovered that these proteins carry built-in signal peptides that direct them into the ER even during their synthesis on cytoplasmic ribosomes and proposed that proteins quite generally have signal sequences that specify their location in the cell, now a basic principle. And, Michael Brown and Joseph Goldstein (in the course of elucidating the mechanism of cholesterol homeostasis; see The discovery of the LDL receptor: Clues to receptor-mediated endocytosis) had just provided the first clear demonstration that selective transport between compartments is mediated by vesicles. They found that transient "coated vesicles," budding from the plasma membrane, carry out the endocytosis of plasma lipoproteins, allowing their cholesterol to be released in lysosomes. These vesicles garner lipoproteins from the medium by means of a receptor localized to the coated regions of membrane involved in budding.

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The sorting problem asks how transport vesicles bud from one compartment and fuse with another one, carrying a chosen set of proteins while leaving others behind. Figuring this out was beyond the reach of microscopy and cell fractionation, which were the main techniques then available to cell biologists but which could reveal neither the underlying protein machinery nor its mechanism. Clearly, new approaches would be needed to accomplish this. One avenue was initiated by Randy Schekman (774), who found ways to select transport mutants of yeast, which permitted protein machinery to be found by molecular cloning of complementing genes when transformation of yeast became possible.

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I chose cell-free reconstitution because it had been the central experimental approach of all biochemistry since the discovery of alcoholic fermentation in yeast extracts by the Buchner brothers at the end of the 19th century. Once reconstituted, cell-free transport could be used as an assay to permit the underlying enzyme proteins to be discovered and purified according to their functional requirements. At the time of our experiment, the mechanisms of ATP synthesis, DNA replication, RNA transcription, protein synthesis, and even the genetic code, were all relatively recent trophies of the reconstitution approach. However, reconstituting intracellular transport seemed especially daunting because membrane compartments are often in intimate proximity in the living cell, and it was then common wisdom that if these spatial relationships were destroyed by homogenization then transport could no longer take place. This strong prejudice — deeply rooted in cell biology from its origin as a branch of microscopic anatomy — no doubt accounted for the skepticism with which our experiment reconstituting transport was greeted for a number of years, until we began to isolate protein machinery.

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The goal of our experiment was to detect transport of a protein between membrane-bound compartments in a cell-free extract. We chose the integral membrane glycoprotein ("G" protein) of vesicular stomatitis virus (VSV), then a favorite for studying membrane biogenesis in animal cells in the days (late 1970s) before it was possible to routinely express cloned genes. G protein is the virus-encoded fusion protein needed for virions to enter and infect cells. It forms the spikes that stud the surface of the virion and is acquired as the virus exits the cell by budding at the plasma membrane. VSV shuts off synthesis of proteins encoded by the host cell and replaces them with its own gene products. G protein is the only protein among them that enters the secretory pathway. The stepwise maturation of G protein's Asn-linked oligosaccharide chains can be conveniently monitored to delineate the progress of G protein within the ER and Golgi because successive maturation steps occur in successive compartments.

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Figure 1  
Processing of N-linked oligo­saccharides in the secretory pathway. N-linked complexes become resistant to cleavage by Endoglycosidase H following addition of N-acetylglucos­amine by GlcNAc transferase I and subsequent release of mannose residues by Golgi mannosidase II in the medial Golgi.

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Figure 2  
Time course of acquisition of Endo-H resistance by VSV G protein. Cells were pulse-labeled with 35S-methionine for 5 minutes and homogenized. Extracts were then incubated without 35S-methionine for the times indicated. After 5 minutes pulse-labeling, the newly synthesized G protein is Endo-H sensitive (compare first two lanes). Between 5 and 10 minutes' incubation in vitro, G protein becomes Endo-H resistant. N and NS are non-secreted VSV proteins. (Adapted from 1887.)

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The pathway by which Asn-linked oligosaccharide chains are matured by processing had then only recently been uncovered by Stuart Kornfeld and others. As shown in Figure 1, it entails the initial addition of a precursor oligosaccharide to the protein in the ER, followed by the sequential removal of certain glucose and mannose residues, and then the addition of the "terminal" sugars N-Acetyl-Glucosamine (GlcNAc), galactose, and sialic acid at successive locations within the Golgi stack. Phillips Robbins had just worked out a neat shortcut for following saccharide processing using SDS protein gels that exploits an unusual microbial endoglycosidase (Endo H) that cleaves the precursor and immature saccharide chains characteristic of the ER and early Golgi, but which cannot cleave processed chains containing GlcNAc or other terminal sugars added later in the Golgi (see Figure 1). Since the saccharide chain (except for the single GlcNAc that is directly linked to Asn) is removed, the overall molecular weight of the glycoprotein is noticeably reduced and its band shifts on an SDS gel (to the GS position; see Figure 2. When only part of the population of G protein has entered and been processed in the Golgi, two bands are observed: the parent band (GR, resistant to EndoH) and the shifted band (GS, sensitive to EndoH). Earlier methods for analysis of saccharide chains, which involved multiple steps of fragmentation and chromatography that required days, were prohibitive for the routine enzyme assay we imagined that reconstitution of transport might become.

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The experiment

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The first attempts at reconstitution began soon after I joined the Stanford biochemistry department in 1978 as an assistant professor. They were carried out by a fearless postdoctoral fellow, Erik Fries, who I was fortunate to attract to the project. Our initial goal was to obtain transport of VSV G proteinfrom the ER to the Golgi in cell-free homogenates. Erik began by radiolabeling VSV-infected hamster cells in tissue culture with 35S-methionine for what we knew would be enough time (about 5 minutes) to allow newly synthesized G protein to enter the ER while hardly entering the Golgi. Then, we disrupted the cells, incubated the homogenate with ATP, and determined whether any of the Endo H-sensitive G protein present at the outset of the cell-free incubation in the ER had become Endo H-resistant (which would indicate transport from the ER to the Golgi).

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Little, if any, GR was produced. When we extended the labeling time, a small signal (GR produced in the homogenate) did appear but it was quickly dwarfed by the ever-increasing amount of GR present in the homogenate at the outset of cell-free incubation due to increasing amounts of G protein entering the Golgi in the cell. No amount of tinkering with the cell-free conditions improved this picture. Worse yet, we could not even be sure that our small signal represented transport taking place in the homogenate.The extra GR produced in vitro could merely have resulted from completion of processing on G protein that had already reached the Golgi before cell disruption.

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Facing this quandary led to the breakthrough. I realized that a mutant hamster cell line (clone 15B) defective in a specific glycosylation step in the Golgi could be harnessed in a variation of the above experiment, both to eliminate the background of Endo H-resistant G protein at the outset of the incubation and to ensure that any glycosylation during the incubation could only result from transport. That mutant, clone 15B, had been isolated by Stuart Kornfeld on the basis of its resistance to an ordinarily toxic plant lectin. It lacks the enzyme N-Acetyl-Glucosamine Transferase I (NAGT-I; also known as GlcNAc transferase), normally found in the central cisternae of the Golgi stack (see Figure 1). As a result of their enzyme deficiency, 15B cells cannot process G protein to Endo H-resistance although they transport the partially processed G protein normally to the cell surface. G protein therefore remains Endo H-sensitive in the ER, Golgi, and in the plasma membrane of 15B cells. So, when homogenates of 35S-methionine-labeled, VSV-infected 15B cells are incubated, the G protein in their membranes will always remain Endo H-sensitive, even if it were to undergo further transport.

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The variation was simply to incubate two homogenates together — one (the "donor") produced from VSV-infected 15B mutant cells, and the other (the "acceptor") produced from uninfected wild-type cells. Now it is possible for Endo H-sensitive G protein (originating in donor 15B membranes) to be processed by NAGT-I (present in acceptor wild-type cell membranes) and thereby be made Endo H-resistant. For example, if vesicles carrying the GS protein were to bud off from donor ER membranes and fuse with the Golgi membranes from the acceptor homogenate, then the transported GS would be converted to GR. A bona fide signal in this revised cell-free reaction explicitly requires that proximity relationships in the cell are not essential for transport, since transport would take place between organelles derived from separate cells.

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With this new design, we could indeed find in vivo labeling conditions that allowed cell-free processing at about the same rate and with about the same efficiency as transport in the cell, as shown in Figure 2. As in the cell, cell-free "transport-coupled glycosylation" is ATP-dependent and occurs between closed membrane-bound compartments, the latter shown by the resistance of the lumenally oriented spike portion of G protein to external proteolytic attack (1887). The first successful in vivo labeling conditions involved a short "pulse"-label with 35S-methionine followed by a 20-minute period of "chase" with unlabeled methionine in the presence of a proton ionophore "uncoupler" that stops transport by inhibiting ATP synthesis in mitochondria. That ATP (or other NTP) is required for transport had been found in 1968 by James Jamieson and George Palade, who used a similar protocol to block exit from the ER at its "transitional elements," specialized regions where vesicles appear to bud off from the Golgi. With this background, the simplest working hypothesis was that we had reconstituted transport from transitional elements of the ER (from 15B cells) to the Golgi (from wild-type cells). However, we also recognized that the identity of the donor compartment was not firmly established as the ER and could be a later compartment (1887).

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Figure 3  
Assay for transport of VSV G protein to the medial Golgi. 15B cells lack GlcNAc transferase I; thus, proteins in these cells never acquire Endo-H resistance, although they are transported through the secretory pathway. To assay for transport, the Golgi-containing fraction from (radiolabeled) VSV-infected 15B cells is incubated with the Golgi-containing fraction from uninfected wild-type cells (plus ATP and cytosol). Acquisition of Endo-H resistance by G protein is a measure of the extent of its transfer from donor compartments to the medial Golgi.

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Indeed, the latter proved to be the case. We subsequently found (1888) that transport could be reconstituted without ATP depletion simply by extending the in vivochase period by enough time (5-10 minutes) to allow the 35S-labeled G protein to reach the 15B cell Golgi before homogenization. This implied that the donor is the Golgi — not the ER or its transitional elements. Since the acceptor is also a Golgi membrane, it followed that transport between two Golgi stacks, one from the 15B cells and the other from the wild-type cells, had been reconstituted (see Figure 3). This was very surprising because it was then textbook knowledge that the Golgi cisternae flow across the stack from its "immature" or "forming" (now termed cis) face to its "mature" (now termed trans) face. This view had been based on anatomical observations rather than functional evidence. [Now, 20 years later, it is clear that transport across the Golgi stack in animals results from a mix of cisternal flow and vesicle transport, the latter being the dominant mechanism for most proteins in most cells and the former important for transport of some particles too large to be accommodated by vesicles (1896)]. The straightforward interpretation of our data was that transfer between Golgi compartments can be mediated by vesicles, and that we had reconstituted this process.

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Figure 4  
Left panel: VSV-infected 15B cells were pulse-labeled with 35S-methionine for 5 minutes, then incubated in medium with unlabeled methionine for 0, 5, 10 or 20 minutes, and homogenized. Extracts were applied directly to the gel. Note that in 15B cells the mature G protein has a slightly lower apparent molecular weight than the immature G protein. L, N, NS and M are non-secreted VSV proteins. Right panel: VSV-infected 15B cells were pulse-labeled, and the radioactivity was chased as indicated for the samples in the left panel. Cells were homogenized, and the Golgi-containing fraction was incubated with the Golgi-containing fraction from uninfected, unlabeled wild-type cells for 0, 20 or 40 minutes, then treated with Endo-H. Note that by 5 minutes chase in vivo and 20 minutes incubation in vitro, Endo-H resistant G protein is detected. (Adapted from 1888.)

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As seen in Figure 4, Endo H-resistance is observed in samples that had been labeled with a 5-minute pulse followed by a 5-minute chase in vivo before homogenization and incubation with acceptor membranes. At this time point, much of the labeled VSV G is known to be present in the Golgi. However, no Endo H resistant G protein is produced in vitro when cells were disrupted right after the 5-minute pulse (no chase); at this time point, the labeled VSV G is still in the ER. With longer times of chase G protein is progressively depleted from the donor Golgi as it transfers to the plasma membrane before cell disruption, and cell-free transport is correspondingly attenuated. Reexamination of our first experiments confirmed that even though energy production was poisoned, this did not occur instantly, and transport had continued during the several minutes required for the cell to use all of its existing ATP. This period was ample to permit much of the G protein to enter the Golgi, thereby reconciling the two experiments. (It now turns out that transport requires only about 10 μM ATP whereas cells normally maintain about 1-3 mM ATP, so transport can continue until the cell has used up most of its ATP.)

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The strong dependence of the efficiency of cell-free transport on the presence of VSV G protein in Golgi versus other membranes convinced us that this was a bona fide reconstitution, and not the result of nonspecific membrane fusion. More detailed analysis (1881; 1882; 1884) soon confirmed this interpretation and provided key improvements. Adding UDP-[3H]GlcNAc (the donor of GlcNAc for glycosylation by NAGT-I) marks each transported G protein with a fixed quantity of radioactivity as it arrives in the acceptor Golgi. Transport is then simply measured by the amount of [3H]-G protein produced. This improvement, made together with William Balch (1881), made it possible to measure initial rates. This, in turn, allowed us to harness the reconstitution as a rapid and routine quantitative assay to guide the purification and discovery of required components.

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Figure 5  
Electron microscope autoradiograph showing that protein containing 3H-GlcNAc is associated with Golgi stacks. The Golgi-containing fraction from VSV-infected 15B cells was incubated with the Golgi-containing fraction from wild-type cells that had taken up UDP- 3H-GlcNAc, and the mixture prepared for electron microscope autoradiography. (Adapted from 1884.)

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Figure 6  
The transport-coupled glycosylation assay was performed with the donor Golgi fraction from VSV-infected 15B cells, and the acceptor Golgi fraction from either rat liver or CHO cells that had taken up UDP- 3H-GlcNAc. The mixtures were then prepared for electron microscope autoradiography. Top panel: The number of Golgi stacks with associated silver grains was counted and plotted as a function of the Golgi stack size. Bottom panel: The number of stacks of each size was counted from Golgi fractions that had not been incubated in the glycosylation assay. The acceptor rat liver Golgi is distinguished by its smaller size from donor 15B (and acceptor CHO cell) Golgi. Since the same 15B Golgi fraction was used as the donor with the different acceptor fractions, we concluded that the difference in Golgi size is due to the acceptor fractions. These results showed that G protein is transported from the mutant 15B Golgi to the rat liver or CHO Golgi containing wild-type GlcNAc transferase I. (Adapted from 1884.)

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Figure 7  
Vesicles bud from Golgi stacks. Left and right panels show electron micrographs of representative donor Golgi fractions incubated with cytosol and ATP for 15 minutes at 0°C (not primed) or at 37°C (primed), respectively. Red arrows indicate apparently budding vesicles. (Adapted from 1881.)

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With 3H to indicate the transported G protein, we could now use autoradiography of electron microscope sections to localize the acceptor site where the labeled protein resides, as shown in Figure 5. This analysis, performed by William Braell (1884), confirmed that the glycosylated G protein resides in morphologically intact Golgi stacks derived from the acceptor homogenate. Thus, the donor- and acceptor-derived Golgi stacks remain as two distinct and unaltered populations, and the processed G protein resides exclusively in the acceptor-derived Golgi population (Figure 6), forcefully implying that G protein is transferred between them by vesicles. We then showed by electron microscopy that 70-90 nm-diameter vesicles containing G protein form at the donor Golgi stacks, as shown in Figure 7 (1882). Later work directly demonstrated that these vesicles are captured by the acceptor stacks and thereby equilibrate between the two populations (1895).

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In retrospect, it is clear that all of the stars were somehow aligned to make this work possible. I came from outside the field of cell biology and therefore had no preconceived notions of what could not be done. The existing background made it clear that reconstitution of transport would be of critical importance. Enough was known to formulate the experiment. And, not trivially, I was fortunate to be in an ideal setting for the type of work involved. The overall environment in the intimate and distinguished Stanford Biochemistry department encouraged persistent risk-taking with the long-range view in mind. In this supportive environment, I never was forced to doubt that we would succeed — eventually. This climate was the result of the dominant presence of Arthur Kornberg, unquestionably the greatest practitioner (and preacher) of enzymology in the second half of the 20th century, and the main reason that I went to Stanford in the first place. My postdoctoral work with Harvey Lodish at MIT taught me how to work with cell-free reactions involving protein synthesis, and introduced me to the VSV system. A collaboration with Phil Robbins at MIT, while in Harvey's lab, made me aware of then-evolving details of oligosaccharide processing, glycosylation mutant cell lines, and of course Endo H. Phil generously donated this then-precious enzyme, and Stuart Kornfeld did the same for 15B cells. Looking back now, I am impressed with the important role an environment can play by encouraging big thinking and supporting junior colleagues who have high ambitions but low budgets. Also critical is the contribution that senior scientists can make by providing key materials with "no strings attached."

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During the two decades since our experiment was published, much progress in our understanding of the Golgi apparatus has been made and it is now apparent that our original reconstitution reproduced a rich array of transport processes that we could not have envisioned at the time. The vesicles we saw budding from Golgi stacks so many years ago (now termed coatomer or COPI-coated vesicles) are actually a mixture of two closely related COPI-vesicles that contain distinct cargo (779). The first population contains the KDEL receptor (see The discovery of the KDEL retrieval signal) but little VSV G protein; it carries retrograde traffic from the Golgi back to the ER. The second kind of COPI vesicle is enriched in cargo like VSV G protein that exit the Golgi at the trans face and also contains small quantities of the resident proteins like glycosyltransferases that are largely restricted to the Golgi stack. Most likely, these vesicles percolate bidirectionally within the stack (1896), although a current alternative view holds that they, like the first population, may move primarily or even exclusively in the retrograde direction. From either perspective, we had obtained a cell-free system that faithfully reconstitutes the kinds of vesicle transport processes that occur in the cell, and which would soon lead us to discover the underlying principles universally used to bud and fuse vesicles.

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The legacy

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Virtually all of the transport pathways linking cytoplasmic membrane compartments to each other and to the cell surface were successfully reconstituted in cell-free extracts of animal or yeast cells in the decade following the experiment (1902), as have numerous other processes that (like vesicle transport) are intimately connected to spatial asymmetry in the cell. This impressive list includes the disassembly and reassembly of the nucleus (1894) and the Golgi (1893) in the cell cycle, the polarized movement of organelles along the cytoskeleton (1897), and the segregation of chromosomes at the mitotic spindle in cell division (1889).

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The cell-free system (and its partial reactions) were utilized as functional assays with which to discover the core protein machinery needed for vesicle budding, capture, and fusion and with which to deduce the molecular mechanisms that are involved (1892):
  • membrane fusion — the ATPase NSF (1883), SNAP proteins (782), and the SNARE complex; and the latter's role in mediating bilayer fusion as well as encoding its compartmental specificity (785).
  • vesicle budding — coatomers (1901) and ARF[GTP] (1898) for Golgi transport vesicles; and the general GTP switch mechanism that triggers budding by coat polymerization (GTP) followed by uncoating (GDP) to permit fusion (1898).
  • vesicle capture/retention — p115 (1900), GM130 and giantin form flexible tethers (1899) that extend from Golgi cisternae to form a matrix that retains COPI vesicles on the stack while still allowing them lateral mobility. This is a variant of the general mechanism that initially captures vesicles for SNARE-dependent fusion at remote acceptor membranes (1892).

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The past few years (1998-2000) have witnessed a satisfying milestone in this body of research with the reductionist demonstration (analogous to the classical proof of structure through synthesis in chemistry) that coatomers, ARFs, and SNAREs provide the crux mechanism of protein transport and sorting in fully defined systems. Coatomers and an ARF[GTP] alone can bud vesicles from pure lipid bilayers (1891); membrane proteins that bind to the coatomers are packaged in the synthetic vesicles and they make budding more efficient by coupling their packaging to coat polymerization (1885). SNARE proteins alone efficiently fuse lipid bilayers (786) with a pattern of specificity that closely reflects the organization of transport pathways among the compartments in the cell (1183). The core principles of budding and fusion are used over and over again in one or another form throughout the cell to create a network that connects the cell's compartments using groups of structurally or functionally homologous proteins that are highly conserved in evolution, so much so that they are frequently interchangeable between species.

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The sorting problem posed by cell biologists a quarter century ago — understanding the mechanism and specificity of protein flow within the cell — has been solved in its principal features. Yet much remains to be learned about how regulatory networks harness the now-familiar engine of core transport machinery to flexibly adjust the pace and direction of membrane flow according to the needs of the cell and tissue to permit an integrated response. Looking ahead, we can expect existing genetic and biochemical approaches combined with new imaging methods to reveal the versatile means by which the principles of vesicle transport are employed in the service of the physiology of the cell, the organ, and the organism in health and in disease.

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The author

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James E. Rothman is Vice Chairman of the Sloan-Kettering Institute of Memorial Sloan-Kettering Cancer Center and founding Chairman of its Cellular Biochemistry and Biophysics Program. Prior to his current appointment, Rothman was the E.R. Squib Professor of Molecular Biology at Princeton University (1988-91) and Professor of Biochemistry at Stanford University School of Medicine (1978-88). He received his B.A. from Yale University in physics (1971), studied medicine at Harvard Medical School (1971-73), received his Ph.D. in biochemistry from Harvard (1976), and was a postdoctoral fellow at MIT (1976-78). Among other honors, Rothman has received Canada's Gairdner Foundation International Award (1996), Saudi Arabia's King Faisal International Prize (1996), the National Academy of Sciences' Lounsbery Award, The Netherlands' Heineken Prize (2000), and Germany's Otto-Warburg Medal (2001). He is a Member of the U.S. National Academy of Sciences (1993) and its Institute of Medicine (1995). His current work centers on the biophysical mechanism and regulation of membrane fusion, and the design of engineered fluorescent indicator proteins for monitoring the activity of signaling and transport pathways in cells and tissues, including those of the nervous system.

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James E. Rothman
Cellular Biochemistry and Biophysics Program
Rockefeller Research Laboratory 517
Memorial Sloan-Kettering Cancer Center
1275 York Avenue, Box 251
New York, NY 10021
Phone: 212 639 8598
Fax: 212 717 3604
E-mail: j-rothman@ski.mskcc.org

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Last Revised on October 12, 2004

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reviews
  • 1892 Mellman, I. and Warren, G. (2000).  The road taken: past and future foundations of membrane traffic.  Cell 100, 99-112.  PubMed   Journal
  • 1896 Pelham, H. R. and Rothman, J. E. (2000).  The debate about transport in the Golgi--two sides of the same coin?  Cell 102, 713-719.  PubMed   Journal
  • 1902 Cook, N. R. and Davidson, H. W. (2001).  in vitro assays of vesicular transport.  Traffic 2, 19-25.  PubMed   Journal
reviews
  • 774 Novick, P., Field, C., and Schekman, R. (1980).  Identification of 23 complementation groups required for posttranslational events in the yeast secretory pathway.  Cell 21, 205-215.  PubMed   Journal
  • 779 Orci, L., Stamnes, M., Ravazzola, M., Amherdt, M., Perrelet, A., Sollner, T.H., and Rothman, J.E. (1997).  Bidirectional transport by distinct populations of COP-I-coated vesicles.  Cell 90, 335-349.  PubMed   Journal
  • 782 Clary, D. O., Griff, I. C., and Rothman, J. E. (1990).  SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast.  Cell 61, 709-21.  PubMed   Journal
  • 785 Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993).  SNAP receptors implicated in vesicle targeting and fusion.  Nature 362, 318-324.  PubMed   Journal
  • 786 Weber, T., Zemelman, B., McNew, J., Westermann, B., Gmachl, M., Parlati, F., Sollner, T.H., Rothman, J.E. (1998).  SNAREpins: minimal machinery for membrane fusion.  Cell 92, 759-772.  PubMed   Journal
  • 1183 McNew, J. A., Parlati, F., Fukuda, R., Johnston, R. J., Paz, K., Paumet, F., Sollner, T. H., and Rothman, J. E. (2000).  Compartmental specificity of cellular membrane fusion encoded in SNARE proteins.  Nature 407, 153-159.  PubMed   Journal
  • 1881 Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984).  Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine.  Cell 39, 405-416.  PubMed   Journal
  • 1882 Balch, W. E., Glick, B. S., and Rothman, J. E. (1984).  Sequential intermediates in the pathway of intercompartmental transport in a cell-free system.  Cell 39, 525-536.  PubMed   Journal
  • 1883 Block, M. R., Glick, B. S., Wilcox, C. A., Wieland, F. T., and Rothman, J. E. (1988).  Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport.  Proc. Natl. Acad. Sci. USA 85, 7852-7856.  PubMed  
  • 1884 Braell, W. A., Balch, W. E., Dobbertin, D. C., and Rothman, J. E. (1984).  The glycoprotein that is transported between successive compartments of the Golgi in a cell-free system resides in stacks of cisternae.  Cell 39, 511-524.  PubMed   Journal
  • 1885 Bremser, M., Nickel, W., Schweikert, M., Ravazzola, M., Amherdt, M., Hughes, C. A., Sollner, T. H., Rothman, J. E., and Wieland, F. T. (1999).  Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors.  Cell 96, 495-506.  PubMed   Journal
  • 1887 Fries, E. and Rothman, J. E. (1980).  Transport of vesicular stomatitis virus glycoprotein in a cell-free extract.  Proc. Natl. Acad. Sci. USA 77, 3870-3874.  PubMed  
  • 1888 Fries, E. and Rothman, J. E. (1981).  Transient activity of Golgi-like membranes as donors of vesicular stomatitis viral glycoprotein in vitro.  J. Cell Biol. 90, 697-704.  PubMed  
  • 1889 Koshland, D. E., Mitchison, T. J., and Kirschner, M. W. (1988).  Polewards chromosome movement driven by microtubule depolymerization in vitro.  Nature 331, 499-504.  PubMed   Journal
  • 1891 Matsuoka, K., Orci, L., Amherdt, M., Bednarek, S. Y., Hamamoto, S., Schekman, R., and Yeung, T. (1998).  COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes.  Cell 93, 263-275.  PubMed   Journal
  • 1893 Misteli, T. and Warren, G. (1994).  COP-coated vesicles are involved in the mitotic fragmentation of Golgi stacks in a cell-free system.  J. Cell Biol. 125, 269-282.  PubMed  
  • 1894 Newport, J. (1987).  Nuclear reconstitution in vitro: stages of assembly around protein-free DNA.  Cell 48, 205-217.  PubMed   Journal
  • 1895 Orci, L., Malhotra, V., Amherdt, M., Serafini, T., and Rothman, J. E. (1989).  Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack.  Cell 56, 357-368.  PubMed   Journal
  • 1897 Schnapp, B. J., Vale, R. D., Sheetz, M. P., and Reese, T. S. (1985).  Single microtubules from squid axoplasm support bidirectional movement of organelles.  Cell 40, 455-462.  PubMed   Journal
  • 1898 Serafini, T., Orci, L., Amherdt, M., Brunner, M., Kahn, R. A., and Rothman, J. E. (1991).  ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein.  Cell 67, 239-253.  PubMed   Journal
  • 1899 Sonnichsen, B., Lowe, M., Levine, T., Jamsa, E., Dirac-Svejstrup, B., and Warren, G. (1998).  A role for giantin in docking COPI vesicles to Golgi membranes.  J. Cell Biol. 140, 1013-1021.  PubMed   Journal
  • 1900 Waters, M. G., Clary, D. O., and Rothman, J. E. (1992).  A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack.  J. Cell Biol. 118, 1015-1026.  PubMed  
  • 1901 Waters, M. G., Serafini, T., and Rothman, J. E. (1991).  'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles.  Nature 349, 248-251.  PubMed   Journal

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