The Road Taken
2000; Cell Press; Volume: 100; Issue: 1 Linguagem: Inglês
10.1016/s0092-8674(00)81687-6
ISSN1097-4172
Autores Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe greatest scientific advance of the last 1000 years was providing the evidence to prove that human beings are independent agents whose lives on earth are neither conferred nor controlled by celestial forces. Although it may be more conventional to measure scientific progress in terms of specific technological developments, nothing was more important than providing the means to release men and women from the hegemony of the supernatural. Establishing human biological autonomy has been slow and is by no means completed. It began with advances in the physical sciences, notably the work of Copernicus and Galileo, who helped to establish that the earth, and thus humankind, did not occupy a unique or privileged position at the center of the universe. Despite the obvious difference in scale, as important were the contributions of the life sciences. Cell biology in particular provided the incontrovertible proof that humans as well as all other living beings consist of individual cells (utilizing the same genetic code) whose activities, inheritance, and ability to assemble into organisms can be understood in logical, if not always straightforward, biochemical terms. This fact demonstrated that however miraculous the existence of life on earth might be, it is not entirely mysterious. Life's mechanisms can be understood, regardless of whether one invokes spiritual or quantum mechanical reasons for its existence. As we stand at the end of the millennium, we probably understand, albeit superficially, nearly all of the basic principles that govern life, death, cognition, and reproduction. We are not yet at the Golden Age predicted some years ago by Gunther Stent (96Stent G The Coming of the Golden Age. Natural History Press, Garden City, NY1969Google Scholar), but it seems increasingly likely that the study of cells as integrated, functional units will be the vehicle that will finally bring us to a complete understanding of our physical existence. Cell biology can be divided into a number of branches, which, with every advance, are becoming progressively intertwined. The study of biological membranes is the branch most responsible for this confluence, largely because it is a cell's system of membranes that provides the very boundaries within which life exists. A membrane separates the panoply of biochemical reactions that define a living cell from the extracellular world. Within the cell, membranes also organize and separate these biochemical reactions from each other, generating the compositionally and morphologically distinct compartments characteristic of eukaryotes. Transfer of material between many of these compartments (in particular, secretory or endocytic compartments) occurs by means of small, membrane-bound vesicles. Although vesicular traffic is extensive, it does not compromise the identity of the compartments involved. Arriving at a general understanding of how these events occur has been a major advance of the past half century. Accordingly, it is appropriate to begin with an appreciation of the intellectual foundations on which our current state of understanding rests. Biology, particularly cell biology, is a quintessentially group effort, with each new concept growing from insights and experimental data contributed by many individual laboratories. Perhaps the reason for this is that single "definitive experiments" are exceedingly rare in cell biology. Progress has been achieved in a graded fashion including influences from ideas and data, which, ironically, may have later proved to be incorrect. It would thus be difficult to enumerate the many individuals responsible for bringing the field to its current state of sophistication. Nevertheless, acknowledging a few people and events is essential to illustrating some key observations and discoveries. The study of membrane traffic dates back to a time before it was clear that membranes even existed. Undoubtedly the most widely known, if not necessarily the most important, observations were provided by the great Italian histologist Camillo Golgi. Golgi developed his silver nitrate–based cytochemical stains to explore the organization of the central nervous system but, in addition, revealed that neurons contain a distinctive internal reticular structure, which has borne his name ever since (31Golgi C Sur la structure des cellules nerveuses.Arch. Ital. Biol. 1898; 30: 60-71Google Scholar). Though the existence of this structure in all eukaryotic cells was hotly debated for the next half century (5Bentivoglio M 1898—the Golgi-apparatus emerges from nerve-cells.Trends Neurosci. 1998; 21: 195-200Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), the "Golgi apparatus" was in fact the first organelle of the secretory or endocytic pathways to be identified. Ironically, Golgi's main conclusion from his studies, namely that the brain consisted of a continuous syncitial network, turned out to be less influential in the long run (37Henry J.M Neurons and nobel-prizes—a centennial history of neuropathology.Neurosurgery. 1998; 42: 143-155Crossref PubMed Scopus (11) Google Scholar). Another critical if less widely appreciated observation of this era was provided by Elie Metchnikoff (62Metchnikoff E Sur la lutte des cellules de l'organisme cintre l'invasion des microbes.Ann. Inst. Pasteur. 1887; 1: 321-326Google Scholar). Born in 1845, just two years after Golgi (he also won his Nobel Prize two years after Golgi), Metchnikoff's discovery of cellular immunity provided a similar cell biological by-product. Whereas the Golgi apparatus was an object without a clear function, the ability of individual cells to internalize extracellular particles by "phagocytosis" suggested a functional intracellular digestive tract. This suggestion was highlighted by the demonstration that ingested particles (e.g., blue litmus) were exposed to acidic pH and presumably degraded following uptake. Metchnikoff provided the first demonstration that cells have internal specializations that carry out specific functions. By the middle of the twentieth century, the advent of electron microscopy (EM) combined with the development of fixatives (e.g., glutaraldehyde [83Sabatini D.D Bensch K Barrnett R.J Cytochemistry and electron microscopy the preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation.J. Cell Biol. 1963; 17: 19-58Crossref PubMed Scopus (1924) Google Scholar]) that permitted the preservation of biological membranes marked the beginning of the modern era of cell biology. The impact was particularly great on the study of membrane traffic, as cells were found to contain an abundance of membrane-bound organelles that soon became associated with the processes of secretion and endocytosis. A great many groups contributed to this effort, but the ones that obviously must be singled out were headed by George E. Palade and Christian de Duve, who made the critical connection between the existence of these structures and individual cell functions (17de Duve C Exploring cells with a centrifuge.Science. 1975; 189: 186-194Crossref PubMed Scopus (174) Google Scholar, 74Palade G Intracellular aspects of the process of protein synthesis.Science. 1975; 189: 347-358Crossref PubMed Scopus (2227) Google Scholar). This connection was aided by the development of cell fractionation and of assays to measure the enzymatic activities associated with subcellular fractions. Palade and de Duve used this approach, combined with EM, to prove the existence in eukaryotic cells of physically distinct organelles that each performed distinct, essential functions. This approach also provided the conceptual and experimental foundation on which virtually every advance in cell biology for the next 50 years was based. Electron Microscopy and Cell Fractionation. It was at this point that the field of membrane traffic was developed, largely due to the efforts of Palade and the impressive "school" of cell biologists he spawned either directly or indirectly. One of the most important experiments early in this period resulted in the functional elucidation of the secretory pathway: that secretory proteins are synthesized in the endoplasmic reticulum (ER), pass through the Golgi complex, and then are packaged into granules for exocytosis at the plasma membrane. The syllogism of ER to Golgi to plasma membrane became cell biology's equivalent of molecular biology's DNA to RNA to protein (both are also not always true!). The secretory pathway's logic was best illustrated by the acinar cell of the exocrine pancreas, among the most professional secretory cells known. Palade and James D. Jamieson made use of the newly developed technique of EM autoradiography, in which newly synthesized secretory proteins were labeled by a pulse of radioactive amino acids and, after various chase periods, detected on EM sections overlaid with a photographic emulsion. Together with biochemical fractionation, these studies definitively demonstrated the initial appearance of secretory proteins over the ER, their transient association with elements of the Golgi complex, their concentration in post-Golgi secretory granules, and their secretagogue-stimulated release from the cell by granule fusion with the plasma membrane (Figure 1). There have been many embellishments of this scenario over the years, but these basic features remain the secretory pathway's most significant elements. Implicit in the elucidation of these events was another fundamental principle of membrane traffic: namely, that transport of secretory proteins between these distinct organelle compartments occurs via vesicular carriers (74Palade G Intracellular aspects of the process of protein synthesis.Science. 1975; 189: 347-358Crossref PubMed Scopus (2227) Google Scholar). In other words, transfer from the ER to the Golgi requires the formation of a transport vesicle by a budding event at the "donor" organelle (ER) and the subsequent fusion of the carrier at the "acceptor" organelle (the Golgi). The importance of this concept cannot be overestimated and is probably the single most important concept underlying the modern understanding of membrane traffic. However, it also created a vexing paradox that has occupied the field ever since. Fluidity, Topology, and Sorting. Concomitant with the explication of the secretory pathway, the nature and properties of biological membranes were also becoming apparent. It was originally thought, based on EM images, that a membrane was a "protein-lipid-protein sandwich." However, the efforts of many groups defined another critical insight: that membranes are lipid bilayers (33Gorter E Grendel F On bimolecular layers of lipoids on the chromocytes of the blood.J. Exp. Med. 1925; 41: 439-443Crossref PubMed Scopus (387) Google Scholar, 22Engelman D.M Lipid bilayer structure in the membrane of Mycoplasma laidlawii.J. Mol. Biol. 1971; 58: 153-165Crossref PubMed Scopus (169) Google Scholar) in which proteins exhibit considerable two-dimensional fluidity (28Frye L.D Edidin M The rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons.J. Cell Sci. 1970; 7: 319-335PubMed Google Scholar). Lipids and transmembrane proteins are generally free to diffuse laterally within the plane of the bilayer, but due to unfavorable energetic considerations, proteins (and many lipids) could not "flip-flop" across the bilayer (13Bretscher M.S Raff M.C Mammalian plasma membranes.Nature. 1975; 258: 43-49Crossref PubMed Scopus (245) Google Scholar). Thus, when two membranes fuse, their sidedness must be maintained: proteins facing the luminal side of an internal organelle or transport vesicle will remain facing the luminal side after budding or fusion. Indeed, the luminal surface of all vesicles and organelles of the secretory (and endocytic) pathway are topologically equivalent to the extracellular environment. Despite the fact that it is now clear that many cell types can organize their membranes into stable or dynamic microdomains, this general view of membranes as a "fluid mosaic" of conserved topology (89Singer S.J Nicolson G.L The fluid mosaic model of the structure of cell membranes. Cell membranes are viewed as two-dimensional solutions of oriented globular proteins and lipids.Science. 1972; 175: 720-731Crossref PubMed Scopus (5581) Google Scholar) remains a foundation of our understanding. However, this view created a problem for rationalizing vesicular transport. Donor and acceptor organelles typically have biochemically distinct membrane compositions. As a result, membrane traffic between them would appear to be an invitation to randomness, an invitation that clearly cannot be accepted. At the conceptual level, this problem is solved by invoking two critical principles, deciphering the mechanisms of which represents a major focus of current effort. First, there is the principle of "molecular sorting," the idea that membrane components are either selectively included within or excluded from nascent transport vesicles. Thus, only those components intended for forward transport need to be sorted from the donor's resident components and removed from the donor organelle. Sorting can occur either by allowing a transported component to interact with one of several known cytoplasmic coat components, by retaining a resident component due to interactions with an intraorganelle or cytoplasmic matrix, or by salvaging those few organellar proteins that inadvertently leave. Second, there is the principle of "vesicle targeting." Vesicles emanating from a donor organelle were long predicted to bear address tags that permit them to interact and fuse with only the appropriate acceptor compartment. These tags are now known to include the organelle-specific family of SNARE proteins and ras-like GTPases of the Rab protein family. Together with proteins that tether vesicles to target membranes, these components help form target-specific protein complexes, which allow for vesicle acceptor compartment recognition and subsequent fusion. Translocation across Membranes. Since protein synthesis (except the protein synthesis that occurs within organelles such as mitochondria and chloroplasts) occurs in the cytosol, it was clear from the outset that secretory and membrane proteins synthesized on cytoplasmic ribosomes somehow gained access to the ER. The mechanism was revealed by experiments showing that all such proteins contain distinctive "signal sequences" that program the energetically unfavorable process of protein translocation across the ER membrane (8Blobel G Dobberstein B Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components.J. Cell Biol. 1975; 67 (a): 852-862Crossref PubMed Scopus (651) Google Scholar, 9Blobel G Dobberstein B Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.J. Cell Biol. 1975; 67 (b): 835-851Crossref PubMed Scopus (1740) Google Scholar, 7Blobel G Intracellular protein topogenesis.Proc. Natl. Acad. Sci. USA. 1980; 77: 1496-1500Crossref PubMed Scopus (866) Google Scholar). Translocation of proteins into the ER of animal cells occurs concomitantly with translation and involves the attachment of polysomes producing a signal sequence–containing protein to the "rough" (or ribosome-studded) regions of the ER. Among the legions of cell biologists who have contributed to this fundamental concept, Günter Blobel—perhaps not surprisingly a direct product of the Palade School—has provided the longest and most influential stream of experimental insight, a view also held by the 1999 Nobel Prize committee. The concept of signal-directed translocation has turned out to be far more robust, flexible, and applicable to a wider range of issues than originally thought. It occurs during secretion in yeast and even in bacteria in ways that are superficially distinct (in the sense that translocation can occur posttranslationally in these organisms) but in fact are remarkably similar in intent and mechanism (51Lyman S.K Schekman R Polypeptide translocation machinery of the yeast endoplasmic reticulum.Experientia. 1996; 52: 1042-1049Crossref PubMed Scopus (15) Google Scholar, 19Duong F Eichler J Price A Leonard M.R Wickner W Biogenesis of the gram-negative bacterial envelope.Cell. 1997; 91: 567-573Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Mitochondria, peroxisomes, and chloroplasts also import nuclear-encoded proteins produced in the cytoplasms of their ancestral hosts, again in a posttranslational fashion (65Neupert W Protein import into mitochondria.Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (944) Google Scholar, 97Subramani S Components involved in peroxisome import, biogenesis, proliferation, turnover, and movement.Physiol. Rev. 1998; 78: 171-188Crossref PubMed Scopus (0) Google Scholar, 45Koehler C.M Merchant S Schatz G How membrane proteins travel across the mitochondrial intermembrane space.Trends Biochem. Sci. 1999; 24: 428-432Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 56May T Soll J Chloroplast precursor protein translocon.FEBS Lett. 1999; 452: 52-56Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Although a decidedly different translocation mechanism is used by these nonvacuolar organelles, the basic logic involving the use of signal sequences to specify entry is preserved. Import (and export) of proteins and nucleic acids into the nucleus uses yet another variation on this theme (32Gorlich D Laskey R.A Roles of importin in nuclear protein import.Cold Spring Harb. Symp. Quant. Biol. 1995; 60: 695-699Crossref PubMed Scopus (15) Google Scholar). In addition to defining a process of fundamental importance, work on translocation had an equivalently strong influence on the development of methods to study membrane traffic. Experiments in which the insertion of proteins into the ER and import into mitochondria were reconstituted represented the first true in vitro reconstitutions of complex activity related to membrane traffic. Such "in vitro assays" facilitated the stepwise dissection and identification of important protein components involved in these processes, components whose physiological relevance was confirmed by subsequent genetic analysis of the same processes in yeast (70Novick P Field C Schekman R Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway.Cell. 1980; 21: 205-215Abstract Full Text PDF PubMed Scopus (1178) Google Scholar). Today, in vitro reconstitution is among the most important and widely used strategies in studying membrane traffic. Now routinely combined with morphological, genetic, and molecular biological approaches, in vitro assays are being applied to increasingly complex problems and, as described below, are allowing further understanding, at the biochemical level, of processes such as membrane fusion, vesicle formation, organelle biogenesis, and protein sorting. Endocytosis and Molecular Sorting. Although endocytosis was the first form of membrane traffic to be appreciated, it did not emerge as a central topic in cell biology until it was suggested as a pathway by which secretory vesicle components were recovered (or "recycled") following their insertion into the plasma membrane during exocytosis (39Heuser J.E Reese T.S Evidence for recycling of synaptic vesicle membrane during neurotransmitter release at the frog neuromuscular junction.J. Cell Biol. 1973; 57: 315-344Crossref PubMed Scopus (1546) Google Scholar). It was the study of endocytosis in nonsecretory cells, however, that established the principle of recycling during membrane traffic. The first indication that membrane components are continuously reutilized for vesicular transport can be traced to the quantitative EM investigations of Zanvil Cohn and Ralph Steinman who showed that, every hour, tissue culture cells internalized amounts of plasma membrane that greatly exceeded their biosynthetic capacity (95Steinman R.M Brodie S.E Cohn Z.A Membrane flow during pinocytosis. A stereologic analysis.J. Cell Biol. 1976; 68: 665-687Crossref PubMed Scopus (390) Google Scholar). Thus, endocytic vesicle components must be recycled back to the plasma membrane for reuse. This was in contrast to the extracellular material internalized as vesicle content, the bulk of which was accumulated intracellularly in lysosomes and degraded. This concept was reinforced and greatly extended by the work of Joseph Goldstein and Michael Brown on the low-density lipoprotein (LDL) receptor (30Goldstein J.L Anderson R.G Brown M.S Coated pits, coated vesicles, and receptor-mediated endocytosis.Nature. 1979; 279: 679-685Crossref PubMed Scopus (1257) Google Scholar). Biochemical techniques and EM were used to analyze defects in LDL uptake or processing exhibited by cells from patients with familial hypercholesterolemia. Not only did this work elucidate the cell biological basis for a major human genetic disorder, but also contributed four basic precepts of membrane traffic: that receptors exist to mediate the intercompartmental transport of specific ligands, that these receptors can be reutilized many times (i.e., recycled), that exposure to acidic pH was a basic mechanism used to dissociate ligand–receptor complexes upon arrival at the appointed destinations, and that receptors (and presumably other membrane proteins) can be selected for specific inclusion in nascent transport vesicles due to the interaction of cytoplasmic tail targeting sequences with cytosolic adaptors. Selectivity in transport involves a unique, tyrosine-containing tetrapeptide sequence that permits the LDL receptor (and many others) to concentrate up to 100-fold at characteristic invaginations of the plasma membrane whose cytoplasmic surfaces were coated with the hexagonal-pentagonal arrays of the protein clathrin (30Goldstein J.L Anderson R.G Brown M.S Coated pits, coated vesicles, and receptor-mediated endocytosis.Nature. 1979; 279: 679-685Crossref PubMed Scopus (1257) Google Scholar, 38Heuser J Evans L Three-dimensional visualization of coated vesicle formation in fibroblasts.J. Cell Biol. 1980; 84: 560-583Crossref PubMed Scopus (339) Google Scholar). These coated pits pinched off to form endocytic coated vesicles, confirming genetically Palade's original predictions concerning the role of such vesicles in mediating macromolecular transport in cells. Clathrin and clathrin-coated vesicles had already been identified in oocytes (81Roth T.F Porter K.R Yolk protein uptake in the oocyte of the mosquito Aedes aegypti L.J. Cell Biol. 1964; 20: 313-332Crossref PubMed Scopus (732) Google Scholar) and later in neurons (39Heuser J.E Reese T.S Evidence for recycling of synaptic vesicle membrane during neurotransmitter release at the frog neuromuscular junction.J. Cell Biol. 1973; 57: 315-344Crossref PubMed Scopus (1546) Google Scholar). These "vesicles in baskets" (44Kanaseki T Kadota K The "vesicle in a basket." A morphological study of the coated vesicle isolated from the nerve endings of the guinea pig brain, with special reference to the mechanism of membrane movements.J. Cell Biol. 1969; 42: 202-220Crossref PubMed Scopus (319) Google Scholar) were presumed to take up nutrients and to recover synaptic vesicle membrane following exocytosis during synaptic transmission. By the mid-1970's, Barbara Pearse had characterized their coat components (75Pearse B.M Coated vesicles from pig brain purification and biochemical characterization.J. Mol. Biol. 1975; 97: 93-98Crossref PubMed Scopus (333) Google Scholar) and in the process provided the first biochemical characterization of a transport vesicle. This was a contribution that qualifies as a "foundation" since it set the standard for work on other transport steps and predated them by over a decade. This work also helped to initiate the incorporation of neurobiology into the study of membrane traffic, an addition that has had a profound effect on both fields. The cell biological analysis of familial hypercholesterolemia was perhaps even more important for the enormous impact it had in shifting the intellectual tradition of cell biology. Together with similar studies on human lysosomal storage diseases (which revealed the critical role of mannose-6-phosphate receptors in targeting acid hydrolases from the Golgi to lysosomes) (46Kornfeld S Mellman I The biogenesis of lysosomes.Annu. Rev. Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1188) Google Scholar), this work completed the addition of genetics into the zeitgeist of mainstream cell biology. It is this change that, more than anything else, marks the transition from the era in which the field was founded to the present and to the future "post-Palade" periods in which genetics, genomics, and molecular biology will dominate the landscape. Just as the previous generation of cell biologists presided over the origin of membrane traffic as a field, the current generation has been responsible for leading the field in a direction where understanding intracellular membrane transport at the molecular and biochemical level has become the predominant consideration. This transition, which began in ∼1980, was characterized by the use of cell culture systems, molecular cloning, enveloped viruses, yeast genetics, and in vitro reconstitution of complex transport events. Although these approaches, at least initially, replaced mammalian tissues as the preferred mode of analysis, they did not supplant the reliance on subcellular fractionation and morphological analysis but rather were added to it. Moreover, these "traditional" strategies evolved in important ways, including the development of immuno-EM of ultrathin cryosections using colloidal gold-coupled protein A and the development of antibody probes as markers for intracellular compartments (91Slot J.W Geuze H.J Immunoelectron microscopic exploration of the Golgi complex.J. Histochem. Cytochem. 1983; 31: 1049-1056Crossref PubMed Scopus (52) Google Scholar). Together, these changes marked the beginning of molecular cell biology, a paradigm shift that affected all aspects of cell biology, particularly of membrane traffic. At the start of the new millennium, we find that most of the major problems identified during the past century, if not solved, at least have logical, biochemically defined frameworks. We now believe we know the fundamental principles underlying how individual membrane components are selectively transferred between organelles by vesicular transport, and how intercompartmental traffic of vesicles occurs without compromising the integrity of the participant organelles. Perhaps the most fundamental aspect of membrane traffic relates to how vesicular carriers identify and fuse with their intended targets. Given the vast array of membrane systems within eukaryotic cells, understanding the specificity of fusion events is critical to understanding membrane traffic and is a difficult challenge. It was long presumed that vesicle–membrane targeting events are controlled by specific interactions of cognate receptor proteins at the cytoplasmic faces of interacting membranes (74Palade G Intracellular aspects of the process of protein synthesis.Science. 1975; 189: 347-358Crossref PubMed Scopus (2227) Google Scholar). Although critical issues remain to be solved, a remarkable synthesis has been achieved based on the convergence of three distinct lines of investigation dating back some 20 years. Reconstitution of Membrane Fusion In Vitro. The first of these story lines begins with the application of enveloped animal viruses to the study of membrane transport (50Lodish H.F Braell W.A Schwartz A.L Strous G.J Zilberstein A Synthesis and assembly of membrane and organelle proteins.Int. Rev. Cytol. Suppl. 1981; 12: 247-307PubMed Google Scholar, 87Simons K Biogenesis of epithelial cell surface polarity.Harvey Lect. 1993; 89: 125-146PubMed Google Scholar). Although professional secretory cells, such as the pancreatic acinar cell, served well for initial descriptions of the secretory pathway, tissues did not lend themselves to the types of manipulations that would be needed to solve questions at the molecular level. Enveloped animal viruses such as vesicular stomatitis virus (VSV), Semliki Forest virus, and influenza virus turn almost any tissue culture cell into a factory committed to the synthesis of viral proteins. Since these viruses express membrane proteins that must be transported to the plasma membrane to permit budding of progeny virions, the infected cells became professional secretory cells for viral envelope glycoproteins. The ability to provide a synchronous pulse of a single type of membrane protein allowed detailed kinetic descriptions of transit through secretory organelles and correlation of their localization (by immuno-EM or cell fractionation) relative to glycosylation state. As a result, transit through the stacked cisternae of the Golgi complex was confirmed to have a distinct polarity, with entry of membrane (and secretory) proteins exported from the ER at the "cis" face and exit at the "trans" face (6Bergmann J.E Tokuyasu K.T Singer S.J Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane.Proc. Natl. Acad. Sci. USA. 1981; 78: 1746-1750Crossref PubMed Scopus (113) Google Scholar). Moreover, two previously unappreciated Golgi compartments were identified. One was the trans-Golgi network (TGN), a system of tubules emanating from the trans-most Golgi cisterna (34Griffiths G Simons K The trans Golgi network sorting at the exit site of the Golgi complex.Science. 1986; 234: 438-443Crossref PubMed Scopus (738) Google Scholar). The TGN proved to be the
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