Epistemic_Statements  

CORD-19:a80bc93a9b4851d9f4925885f9436f3739781333 JSONTXT 9 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T1 350-532 Epistemic_statement denotes This period of advance has largely been due to the Idevelopment of powerful new biochemical, morphological, #and genetic approaches to unraveling the complexities of 'this organelle.
T2 533-634 Epistemic_statement denotes While much remains to be discovered, the Iproblem now is how to integrate this wealth of information.
T3 635-808 Epistemic_statement denotes 'To see if this is possible, we will first summarize how the lslolgi is commonly believed to work and then evaluate the lstrength of the evidence that underlies these views.
T4 1051-1233 Epistemic_statement denotes This period of advance has largely been due to the Idevelopment of powerful new biochemical, morphological, #and genetic approaches to unraveling the complexities of 'this organelle.
T5 1234-1335 Epistemic_statement denotes While much remains to be discovered, the Iproblem now is how to integrate this wealth of information.
T6 1336-1509 Epistemic_statement denotes 'To see if this is possible, we will first summarize how the lslolgi is commonly believed to work and then evaluate the lstrength of the evidence that underlies these views.
T7 1510-1593 Epistemic_statement denotes Present View of the Golgi 'The Golgi complex is essentially a carbohydrate factory.
T8 2348-2505 Epistemic_statement denotes The cisternae are also associated with an #array of small vesicles, as well as with a network of tubules (emanating from both sides of the stack (Figure 1 ).
T9 3267-3571 Epistemic_statement denotes Given the directionality of transport and the sequential nature of the glycosylation events, it is commonly thought that the Golgi consists of a series of functionally distinct compartmentscorresponding to structurally distinct cisternae or sets of cisternae (Dunphy et al., 1985; Duden et al., 1991 a) .
T10 3817-4016 Epistemic_statement denotes Enzymes involved in intermediate steps, such as the transfer of N-acetylglucosamine to the mans sugars (GlcNAc transferase I), are thought to be restricted to medial cisternae (Dunphy et al., 1985) .
T11 4329-4510 Epistemic_statement denotes The scheme thus predicts that a series of sequential biochemical reactions occurs within separate compartments, through which glycoprotein substrates pass as if on an assembly line.
T12 4778-4923 Epistemic_statement denotes Such a mechanism is consistent with the existence of the numerous small vesicles found in the vicinity of the Golgi (Farquhar and Palade, 1981) .
T13 4924-5156 Epistemic_statement denotes Since transport of proteins and small molecules through the secretory pathway does not appear to require any identifiable signals, it is also thought that transit through the Golgi occurs by "bulk flow" (Pfeffer and Rothman, 1987) .
T14 5157-5273 Epistemic_statement denotes In other words, the intercisternal transport vesicles may not selectively include components intended for transport.
T15 5274-5488 Epistemic_statement denotes Rather, resident proteins of any one cisterna must be selectively excluded from entering these transport vesicles, most likely reflecting the selective retention of resident proteins in their appropriate cisternae.
T16 5489-5711 Epistemic_statement denotes A few cellular and viral glycoproteins are known that appear to have such retention signals, resulting in their accumulation in the Golgi (Colley et al., 1989; Swift and Machamer, 1991; Nilsson et al., 1991; Munro, 1991) .
T17 6655-7114 Epistemic_statement denotes In the TGN of polarized or secretory cells (endocrine and exocrine secretory cells, epithelial cells, and neurons), other routes of exit may occur, leading to inclusion either within storage granules (Kelly, 1985; Huttner and Tooze, 1989) or within transport vesicles targeted specifically to the apical or basolateral plasma membranes (Bennett et al., 1988; Wandinger-Ness et al., 1990) or to the dendritic and axonal cell surfaces (Dotti and Simons, 1990) .
T18 7115-7174 Epistemic_statement denotes How Good Is the Evidence for the Current View of the Golgi?
T19 7700-7842 Epistemic_statement denotes Although originally thought to be a "fixation artifact," the basic cisternal organization of the Golgi complex is now accepted as a certainty.
T20 8188-8293 Epistemic_statement denotes What is less than clear, however, is the extent to which the Golgi is divided into distinct compartments.
T21 8658-8812 Epistemic_statement denotes However, a definitive demonstration that these enzymes exist in separate cisternae can only be provided by visualizing two or more enzymes simultaneously.
T22 8813-9004 Epistemic_statement denotes The Golgi complex in the cell is not the idealized stack that we are accustomed to recognize in diagrams, but it may be subject to twists that make individual sections difficult to interpret.
T23 9005-9089 Epistemic_statement denotes To date, simultaneous localization of two or more enzymes has not been accomplished.
T24 9090-9339 Epistemic_statement denotes In addition, the localization of key enzymes such as mannosidase I remains to be established; this is a critical unknown, since mannosidase I is thought to be the first glycosidase to modify N-linked oligosaccharides upon their arrival in the Golgi.
T25 9340-9456 Epistemic_statement denotes A further complication is introduced by the fact that there appears to be considerable variation between cell types.
T26 9693-9837 Epistemic_statement denotes In neighboring absorptive cells of the gut, however, almost lhe entire Golgi stack is labeled (except forthe cis cisterna) (Roth et al., 1986) .
T27 9838-9982 Epistemic_statement denotes It has also recently become clear that Golgi glycosyltransferases may have multiple isoforms with either Golgi or plasma membrane distributions.
T28 10258-10552 Epistemic_statement denotes Cell fractionation experiments have also contributed to our view of the Golgi as a multicompartment organelle, since it has been possible to partly separate membranes containing several of the enzymes involved in Golgi processing events (Goldberg and Kornfeld, 1983; Dunphy and Hothman, 1983) .
T29 10553-10618 Epistemic_statement denotes However, interpretation of these data is limited on two accounts.
T30 10619-10787 Epistemic_statement denotes First, in the absence of definitive immunocytochemical localization, it is impossible to know whether the different fractions correspond to different sets of cisternae.
T31 11376-11607 Epistemic_statement denotes Thus, although consistent with the existence of at least three physically and functionally distinct compartments, these observations also suggest that many of the carbohydrate processing steps may occur within a single compartment.
T32 11949-12179 Epistemic_statement denotes Indeed, having GlcNAc transferase and galactosyltransferase in a common compartment would explain the existence of lactosamine repeats in the terminal chains of many glycoproteins (Cummings and Kornfeld, 1984; Howe et al., 1988) .
T33 12180-12463 Epistemic_statement denotes A further reason for caution in interpreting existing imlmunocytochemistry and fractionation results is the growing appreciation of the fact that the individual Golgi cister-Inae-and perhaps even entire stacks-may not be as datic and structurally distinct as we have come to believe.
T34 12464-12660 Epistemic_statement denotes IExamination of the Golgi using high voltage electron milcroscopy has suggested that adjacent stacks of cisternae (are joined via extensive tubular interconnections (Rambourg and Clermont, 1990) .
T35 12661-12932 Epistemic_statement denotes Similarly, real-time imaging of Iliving cells labeled with the fluorescent lipid analog NBD-Iceramide has revealed that the Golgi is capable of forming ,a large number of tubules that may serve to connect other-'wise "separate" stacks of cisternae (Cooper et al., 1990) .
T36 12933-13083 Epistemic_statement denotes Whether the tubules form connections between cisternae in register (i.e., cis to cis, medial to medial) or out of register (cis to medial) is unknown.
T37 13084-13244 Epistemic_statement denotes The possibility must also be considered that even within a single stack, adjacent cisternae might at least transiently possess similar tubular interconnections.
T38 13253-13355 Epistemic_statement denotes A question that clearly needs discussion at this point is what exactly defines a cellular compartment.
T39 13356-13541 Epistemic_statement denotes Classically, membrane-bounded compartments have been viewed as comprising physically distinct entities each with its own distinct protein compositions and, presumably, unique functions.
T40 13542-13692 Epistemic_statement denotes Accordingly, all of any one type of organelle can be considered a compartment, but so can a distinct region (or "subcompartment") within an organelle.
T41 13693-13834 Epistemic_statement denotes By this criterion, compartment boundaries should be easily delineated by immunoelectron microscopic localization of resident marker proteins.
T42 13835-13895 Epistemic_statement denotes However, this simple view is complicated by several factors.
T43 13896-14111 Epistemic_statement denotes For example, even the localization of two Golgi enzymes to different cisternae by double label immunoelectron microscopy might not unequivocally demonstrate that they are present in physically distinct compartments.
T44 14112-14309 Epistemic_statement denotes Such a distribution of enzymes could reflect the existence of subdomains in an otherwise continuous membrane array, analogous to the lateral heterogeneity found between the rough and the smooth ER.
T45 14310-14512 Epistemic_statement denotes Conversely, markers may be segregated into physically distinct membranes that nevertheless remain functionally continuous by repeated membrane fissions and fusions or transient tubular interconnections.
T46 14513-14700 Epistemic_statement denotes The existence of such homotypic interactions-interactions among "like" elements-would play an important role in preserving continuity among equivalent but physically separated organelles.
T47 14835-14960 Epistemic_statement denotes The definition of a compartment is further confounded when considering organelles that are also involved in membrane traffic.
T48 14961-15181 Epistemic_statement denotes Given that there is likely to be continuous transport and recycling of membrane components between such compartments, compartment identities are not static and therefore may not always be entirely biochemically distinct.
T49 15291-15480 Epistemic_statement denotes Conceivably, endogenous (or resident) components may be defined by their ability to interact with a compartment-specific structural framework, such as a cytosolic membrane coat or skeleton.
T50 15481-15684 Epistemic_statement denotes It is clear that only by identifying the machinery responsible for entry into, retention within, or exit from a compartment will we be able to precisely and consistently determine compartment boundaries.
T51 15685-15950 Epistemic_statement denotes In the absence of this information, compartments can only be tentatively defined as functionally equivalent and possibly physically continuous populations of membrane-bounded structures that are both functionally and physically distinct from all other compartments.
T52 16176-16405 Epistemic_statement denotes scribe definitively the compartmentalization of the Golgi complex, models for the organization of the Golgi should be kept as simple as possible, adding complexity only as it is necessitated by additional functional requirements.
T53 16975-17276 Epistemic_statement denotes Although the details of Golgi morphology vary considerably between different cell types, such a three compartment subdivision is generally consistent with the overall structural organization of the mammalian Golgi from threedimensional reconstructions (Rambourg and Clermont, 1990) (Figures 2 and 3) .
T54 17277-17525 Epistemic_statement denotes It is also consistent with the functional organization of the Golgi in Saccharomyces cerevisiae, which, on the basis of mutations affecting Golgi function, has recently been proposed toconsist of at least three compartments (Graham and Emr, 1991) .
T55 17652-17896 Epistemic_statement denotes Structurally, however, the cis-most cisterna of the Golgi is typically associated with a continuous array of tubules that are selectively stained after prolonged osmication (Lindsey and Ellisman, 1985; Rambourg and Clermont, 1990) ( Figure 2 ).
T56 17897-18104 Epistemic_statement denotes Given this distinctive morphology, and by analogy to the TGN, it seems appropriate to refer to this region as a distinct compartment called the CGN (Huttner and Tooze, 1989; Hsu et al., 1991; Pelham, 1991) .
T57 18105-18369 Epistemic_statement denotes For several reasons, it also seems reasonable to suggest that the CGN also includes the poorly understood but possibly interconnected system of tubules between the ER and the Golgi, referred to as the "salvage compartment" (Warren, 1987; Pelham, 1991) (Figure 3 ).
T58 18370-18597 Epistemic_statement denotes In addition to serving as a site of entry into the Golgi, recent evidence suggests that the CGN functions in the recycling of protein and lipid components back to the ER, while having a relatively limited role in glycosylation.
T59 19086-19231 Epistemic_statement denotes The lateral arrows would be sites where direct fusions would interconnect CGN-CGN, medialmedial, and TGN-TGN compartments of two adjacent stacks.
T60 19467-19650 Epistemic_statement denotes In animal cells, the lysosomal enzyme cathepsin D was given a C-terminal KDEL tag and found not to receive any terminal sugar modifications associated with Golgi-linked glycosylation.
T61 19651-19722 Epistemic_statement denotes However, mannose residues on the enzyme did receive a GlcNAc-phosphate.
T62 19723-19919 Epistemic_statement denotes Thus, although the KDEL clonstruct reached a GlcNAc-phosphotransferase-containing compartment, it did not encounter mannosidase I, a glycosidase that would remove the phosphotransferase substrate.
T63 19920-20211 Epistemic_statement denotes Although it may yet turn out that some KDEL proteins receive further oligosaccharide modifications, K.DEL receptors can retrieve these proteins before they reach the bulk of the oligosaccharide transferases; thus, recycling is a function associated with a distinct "early" Golgi compartment.
T64 20212-20385 Epistemic_statement denotes Without immunocytochemical localization of these constructs (the KDEL receptor or the phosphotransferase), it is impossible to be certain that these events occur in the CGN.
T65 20386-20695 Epistemic_statement denotes However, this appears likely, given that our model assumes that the tubules and vesicles defining the CGN are also associated with antigens such as ~53, ~58, and mb2 (Saraste et al., 1987; Schweizer et al., 1988; Chavrier et al., 1990) which have been used to define the so-called "intermediate compartment" .
T66 20696-20906 Epistemic_statement denotes These antigens label structures that appear to serve as intermediates in transport from the ER to the Golgi as well a.s in the recycling of Golgi components back to the ER (I-ippincott- Schwartz et al., 1990) .
T67 20907-21186 Epistemic_statement denotes The markers also label presumed sites of exit from the ER ("transitional elements," Palade, 1975) as well as the cis-most Golgi cisterna (Saraste and Svensson, 1991; Lotti et al., 1992) , suggesting that they are not endogenous CGN components, but recycle between the ER and CGN.
T68 21663-21784 Epistemic_statement denotes We thus view the CGN as a unique Golgi compartment responsible for receiving and sorting components arriving from the ER.
T69 21785-21994 Epistemic_statement denotes Structurally, we predict it will ordinarily (but perhaps not always) consist of the cis-most Golgi cisterna and the associated vesicles and tubules that include the intermediate compartment of Schweizer et al.
T70 22310-22601 Epistemic_statement denotes These would include the addition of the first N-acetylgalactosamine residue to O-linked sugars, the assembly of coronavirus particles (Tooze et al., 1988; Machamer et al., 1990) and fatty acylation of membrane protein cytoplasmic domains (Rizzolo and Kornfeld, 1988; E3onatti et al., 1989) .
T71 22750-22818 Epistemic_statement denotes The medial Golgi would function primarily as a glycosylation device.
T72 23002-23231 Epistemic_statement denotes While distinct from the CGN or TGN, we assume that the medial cisternae represent a single functionally continuous compartment, irrespective of the actual number of physically separate cisternal elements in any one Golgi complex.
T73 23232-23337 Epistemic_statement denotes Nevertheless, owing to the sidedness of the Golgi, the medial cisternae may appear structurally distinct.
T74 23338-23566 Epistemic_statement denotes For example, since one face of a medial cisterna is apposed to the CGN while another face is apposed to the TGN, the two faces may have different compositions, owing to their interactions with structurally distinct compartments.
T75 23567-23779 Epistemic_statement denotes Most of the glycosylation reactions (N-linked, O-linked, as well as glycolipid synthesis), aswell as polysaccharide synthesis normally associated with the Golgi, are viewed as occurring in the medial compartment.
T76 23780-23926 Epistemic_statement denotes This arrangement would account for cell type heterogeneity in cisternal number and enzyme distribution by placing them all in asingle compartment.
T77 24002-24114 Epistemic_statement denotes If the medial Golgi serves only to mediate glycosylation reactions, why the characteristic cisternal morphology?
T78 24115-24190 Epistemic_statement denotes One possibility might be simply to enhance the efficiency of glycosylation.
T79 24191-24348 Epistemic_statement denotes Although precise measurements are difficult to make, transport systems for sugar nucleotide precursors are kinetically inefficient (Deutscher et al., 1984) .
T80 24349-24603 Epistemic_statement denotes Moreover, while the terminal transferases also have relatively high K,s for substrate (high micromolar range), they may work at rates that may exceed the rate of sugar nucleotide transport (Weinstein et al., 1982; C. Hirschberg, personal communication) .
T81 24604-24776 Epistemic_statement denotes Thus, the cisternal stack by its flattened structure may serve to enhance the efficiency of glycosylation by minimizing lumenal volume and increasing membrane surface area.
T82 24883-25091 Epistemic_statement denotes Structurally, it is likely to be defined by the sacculotubular network located on the trans side of the Golgi that varies in structure in different cell types (Rambourg and Clermont, 1990) (Figures 2 and 3) .
T83 25351-25456 Epistemic_statement denotes The TGN may also have a lower pH than preceding compartments in the pathway (Anderson and Pathak, 1985) .
T84 25802-25953 Epistemic_statement denotes The extreme case is represented by the Golgi complex of algae where flakes are assembled in the lumen of the Golgi cisternae (Melkonian et al., 1991) .
T85 25954-26206 Epistemic_statement denotes Since these large flakes are first observed several cisternae away from the cis side, it seems reasonable to assume that the flakes assemble in saccular portions of the TGN and that entire TGN cisternae peel off and are transported to the cell surface.
T86 27124-27275 Epistemic_statement denotes We propose that the TGN also operates as a directional valve to control the flow of membrane proteins and lipids in biosynthetic transport (Figure 3) .
T87 27276-27450 Epistemic_statement denotes Such a function would contribute to the efficiency of forward transport by preventing passenger proteins and lipids from returning to the medial Golgi after reaching the TGN.
T88 27451-27543 Epistemic_statement denotes In practice, this would predict that there is no recycling from the TGN to the medial Golgi.
T89 27675-27850 Epistemic_statement denotes Several lipids such as cholesterol, glycolipids, and sphingomyelin are present in very low concentrations in the ER, although they are abundant in the Golgi (van Meer, 1989) .
T90 27851-27986 Epistemic_statement denotes If there were lipid recycling from each Golgi compartment, it would be difficult to account for these differences in lipid composition.
T91 28067-28279 Epistemic_statement denotes Several studies have demonstrated directly that there is little backflow of glycoproteins from the TGN into proximal Golgi compartments (Duncan and Kornfeld, 1988; Neefjes et al., 1988; Chege and Pfeffer, 1990) .
T92 28280-28552 Epistemic_statement denotes Also, recycling of glycosphingolipids through the TGN (Schwartzmann and Sandhoff, 1990) would be easier to explain if these plasma membrane lipids would not encouter medial Golgi enzymes; otherwise, with time, only complex glycolipids would be present on the cell surface.
T93 28614-28765 Epistemic_statement denotes The second key element underlying our current view of the Golgi concerns the role of carrier vesicles in mediating transport through the Golgi complex.
T94 28766-28972 Epistemic_statement denotes While transport vesicles may represent the most likely mechanism to accomplish transfer between compartments and/or individual cisternae, how strong is the evidence that they actually perform this function?
T95 28973-29141 Epistemic_statement denotes In intact cells, perhaps the best argument comes from the early cell-cell fusion experiments of Rothman and colleagues (Rothman et al., 1984a (Rothman et al., , 1984b .
T96 29240-29507 Epistemic_statement denotes Transfer of the VSV G protein from Golgi membranes in the infected to the uninfected cells was monitored by a glycosylation event that could be carried out by only the uninfected recipient, which was chosen to complement the glycosylation defect of the infected cell.
T97 29859-29967 Epistemic_statement denotes Conceivably, the same transport vesicles could mediate intercisternal transport within a single Golgi stack.
T98 29968-30029 Epistemic_statement denotes The major limitation of these data is that they are indirect.
T99 30119-30308 Epistemic_statement denotes One cannot exclude the possibility that the G protein moved by lateral diffusion through tubules connecting the heterologous Golgi stacks, which otherwise remain separated during the assay.
T100 30784-30918 Epistemic_statement denotes That such transport can be achieved has been demonstrated over the past several years by the pioneering work of Rothman and coworkers.
T101 31396-31669 Epistemic_statement denotes This transfer has also been dissected kinetically into two major stages: an early stage presumed to correlate with the budding of transport vesicles from the donor Golgi, and a late stage reflecting the docking and subsequent fusion of the vesicles with the acceptor Golgi.
T102 32167-32212 Epistemic_statement denotes However, the identi-DONOR ACCEPTOR Fllgure 4.
T103 32441-32599 Epistemic_statement denotes (ES) Vesicle-independent direct fusions between donor and acceptor compartments (CGN-CGN, medial-medial, and TGN-TGN), perhaps mediated by tubular extensions.
T104 32600-32644 Epistemic_statement denotes While these tubules may be amplified by BFA.
T105 32645-32731 Epistemic_statement denotes as described in the text, they may exist and function even in the absence of the drug.
T106 32732-33017 Epistemic_statement denotes ties of the donor and acceptor cisternae have not yet been established, nor can kinetic analysis alone provide a unique interpretation, especially when transport is monitored indirectly by linkage to glycosylation, which itself is a kinetically complex series of reactions (see above).
T107 33376-33751 Epistemic_statement denotes One important factor, designated NSF (NEM-.sensitive fusion protein), is homologous to the yeast sec78 gene product and is thought to act at a late stage in transport, at the time when transport vesicles fuse with the acceptor membranes Orci et al., 1989; Rexach and Schekman, 1991) both in ER to Golgi and in Golgi as well as in post-Golgi transport (Graham and Emr, 1991) .
T108 33752-33806 Epistemic_statement denotes The precise function of NSF, however, remains unknown.
T109 33807-34003 Epistemic_statement denotes Another factor, designated SNAP (for soluble NSF attachment protein), is homologous to the yeast sec77 gene product and is thought to be involved in NSF binding to membranes (Clary et al., 1990) .
T110 34004-34206 Epistemic_statement denotes The strongest evidence for the existence of a vesicular intermediate in the in vitro transport reaction has been provided by the identification and isolation of the presumed carrier vesicles themselves.
T111 34517-34722 Epistemic_statement denotes One of these, B-COP, was previously identified by Kreis and coworkers as a 110 kd peripheral protein tightly associated with the Golgi (Allan and IKreis, 1986; Duden et al., 1991b; Serafini et al., 1991) .
T112 34723-34800 Epistemic_statement denotes These non-clathrin-coated vesicles are good candidates ,for carrier vesicles.
T113 35036-35274 Epistemic_statement denotes Most interestingly, treatment of cells or isolated membranes with the nonhydrolyzable GTP analog, GTPyS, both blocks glycosylation of the VSV G protein and results in the accumulation of VSV G protein-containing coated buds and vesicles .
T114 35385-35469 Epistemic_statement denotes The stage at which these inhibitors act remains unknown (Donaldson et al., 1991 b) .
T115 35470-35694 Epistemic_statement denotes In spite of this large amount of correlative evidence, additional data will be required before it is certain that the coated vesicles serve as unique and obligatory transport intermediates either in vitro or in intact cells.
T116 35695-35853 Epistemic_statement denotes Perhaps the most important piece of missing information pertains to whether the vesicles have a composition consistent with their presumed transport function.
T117 36328-36549 Epistemic_statement denotes Another critical unknown is a demonstration that the Golgi coated vesicles are functional in vitro, i.e., that VSV G protein-containing vesicle fractions can be used to reconstitute transport when added to acceptor Golgi.
T118 36763-36816 Epistemic_statement denotes Thus, in principle, this approach should be possible.
T119 36817-36986 Epistemic_statement denotes However, with all such experiments, it will be important to ensure that the coated vesicle fractions used are not contaminated with uncoated, fusion-competent membranes.
T120 36987-37273 Epistemic_statement denotes BFA Blocks Vesicle Formation without Blocking Transport A reason for examining the features of the Golgi vesicles so carefully is indicated by a recent paper that raises the possibility that, at least under certain conditions, these vesicles may not be required for transport in vitro .
T121 37704-37899 Epistemic_statement denotes The mechanism of BFA action is unknown, but one intriguing property is its ability to rapidly, and reversibly, trigger the dissociation of B-COP (Donaldson et al., 1991a; Klausner et al., 1992) .
T122 37900-38039 Epistemic_statement denotes When isolated Golgi was treated with BFA, P-COP was no longer associated with the membranes nor were any coated buds or vesicles detected .
T123 38498-38665 Epistemic_statement denotes Alternatively, BFA may induce an aberrant form of transport that nevertheless utilizes enzymatic machinery required for vesicular transport in the absence of the drug.
T124 38666-38741 Epistemic_statement denotes It is at present impossible to distinguish between these two possibilities.
T125 38742-38949 Epistemic_statement denotes However, BFA-treated Golgi in vitro appear to form tubular extensions that may serve to interconnect donor and acceptor Golgi stacks, thus resulting in transfer to the acceptor across these tubular bridges .
T126 38950-39064 Epistemic_statement denotes This possibility is appealing since it could reflect the BFA-induced tubulation of the Golgi seen in intact cells.
T127 39065-39229 Epistemic_statement denotes However, additional electron microscope immunocytochemistry will be required to establish continuous interconnections between antigenically distinct Golgi elements.
T128 39267-39470 Epistemic_statement denotes The fact that "transport" (i.e., G protein glycosylation) can occur in the absence of vesicle formation raises a number of important questions: What transport step(s) is actually being measured in vitro?
T129 39471-39564 Epistemic_statement denotes What is the relationship of the events reconstituted in vitro to those found in intact cells?
T130 39565-39662 Epistemic_statement denotes Is transport in vitro ever completely dependent on vesicle formation, even in the absence of BFA?
T131 39663-39792 Epistemic_statement denotes If vesicles are not involved, is transfer from donor to acceptor Golgi mediated by direct fusion, perhaps via tubular extensions?
T132 39793-40000 Epistemic_statement denotes As mentioned above, it is unclear whether the G protein is transferred across a predicted compartment boundary (donor CGN to acceptor medial) or within a single compartment (donor medial to acceptor medial).
T133 40001-40163 Epistemic_statement denotes It is becoming increasingly clear that all membranebound organelles have a propensity to form tubules in intact cells and in vitro, with or without BFA treatment.
T134 40681-40809 Epistemic_statement denotes Cell-free assays have documented that the early endosome elements can fuse avidly with each other (Gruenberg and Howell, 1989) .
T135 40992-41215 Epistemic_statement denotes The Golgi complex is also a dynamic structure that can be disassembled by microtubule depolymerization into fragments that remain functional despite being dispersed throughout the cytoplasm (Thyberg and Moskalewski, 1985) .
T136 41359-41507 Epistemic_statement denotes A similar series of events must occur during mitosis when the Golgi fragments into populations of small vesicles and tubules (Lucocq et al., 1989) .
T137 41554-41837 Epistemic_statement denotes Given that homotypic fusion occurs so commonly, it would be premature to dismiss the possibility that the intra-Golgi transport activity observed in vitro actually reflects a process of regulated tubule formation and/or direct fusion of donor and acceptor Golgi elements (Figure 4) .
T138 41838-42084 Epistemic_statement denotes Indeed, even in untreated Golgi, both in vitro and in intact cells, tubules can be found that are similar to those observed in BFA-treated Golgi (Hermo et al., 1980; Braell et al., 1984 [see Figure 8b in this reference]; Griffiths et al., 1985) .
T139 42085-42315 Epistemic_statement denotes That a direct fusion event is possible is also suggested by the fact that the predicted diffusion coefficient of intact Golgi stacks is ~1 O-fold less than that of the much smaller coated vesicles (N. Ktistakis, unpublished data).
T140 42316-42575 Epistemic_statement denotes Thus, one might expect a decrease in the kinetics of VSV G protein transfer if BFA were to switch the signal generated in vitro from a vesicle-dependent phenomenon (as is presumed to occur in the absence of the drug) to a direct, tubule-mediated fusion event.
T141 42576-42691 Epistemic_statement denotes However, G protein is transported from donor to acceptor with the same kinetics in the presence or absence of BFA .
T142 42692-43014 Epistemic_statement denotes Finally, the fact that the two N-linked chains of VSV G protein are processed simultaneously in vitro as opposed to sequentially in permeabilized cells may also indicate that compartmental boundaries may not be strictly preserved, i.e., that direct fusion may occur, during the cell-free assay (Schwaninger et al., 1991) .
T143 43015-43219 Epistemic_statement denotes The minimal model that we discussed (see Figure 3 ) predicts only two functional and presumably physical discontinuities in theGolgi: from theCGN to the medial Golgi, and from the medial Golgi to the TGN.
T144 43450-43868 Epistemic_statement denotes Considering available data, we consider it likely that the intercompartmental transfers between CGNlmedial and medial/TGN boundaries occur in vivo via vesicular carriers While the putative transporters remain to be identified, the P-COP-containing vesicles Serafini et al., 1991) are obvious but unconfirmed candidates, whether or not they are ultimately found to function as carrier vesicles in in vitro Golgi assays.
T145 43869-44183 Epistemic_statement denotes The tubular interconnections known to occur in the Golgi may also play a role in intercompartmental transfer, but we view these as probably being more important for establishing and maintaining intracompartmental links that serve to interconnect functionally identical Golgi compartments and to mix their contents.
T146 44184-44345 Epistemic_statement denotes These tubular connections may break and fuse continuously, accounting for the dynamic behavior of the Golgi complex after microtubule disassembly and reassembly.
T147 44346-44550 Epistemic_statement denotes However, if their formation is tightly controlled by a regulated process of assembly and disassembly of the f&COP-containing Golgi coat, even tubule-based intercompartmental transfer is not inconceivable.
T148 44551-44737 Epistemic_statement denotes Moreover, whether cell-free assays of Golgi function reflect such an intercompartmental transfer, or intracompartmental transfer between wild-type and mutant Golgi, remains to be solved.
T149 44738-44867 Epistemic_statement denotes Only further characterization of the machinery responsible for intra-Golgi transport will decide between these two possibilities.
T150 44868-45159 Epistemic_statement denotes Nevertheless, based on the evidence both from the in vitro assays and from yeast genetics, it is clear that the few proteins identified thus far, such as NSF and SNAP, are generally important elements, irrespective of the precise pathways or whether transport occurs via vesicles or tubules.
T151 45557-45746 Epistemic_statement denotes The evidence for this view is largely indirect or negative, in that efforts to identify discrete signals on proteins that are required for forward transport have thus far been unsuccessful.
T152 46047-46242 Epistemic_statement denotes If this view is correct, selectivity in I.ransport through the secretory pathway may occur by "default," i.e., transport of passenger proteins proceeds owing to the absence of a retention signal.
T153 46243-46360 Epistemic_statement denotes This concept, however, is not necessarily incompatible with the existence of selective signals for forward transport.
T154 46361-46483 Epistemic_statement denotes It is clear that such signals exist and play an important role in directing the traffic of proteins as they leave the TGN.
T155 46797-47035 Epistemic_statement denotes Since the transport of ,fluorescent lipids from the Golgi to the plasma membrane Iin nonpolarized cells occurs very rapidly, it is thought that "constitutive" transport from the Golgi to the cell surface may not require specific signals .
T156 47251-47434 Epistemic_statement denotes While these experiments suggest that signals are not necessary for transport, the fact that they are released with rapid kinetics does not alone establish the absence of such signals.
T157 47435-47652 Epistemic_statement denotes Experiments showing that different secretory and membrane proteins are transported with different kinetics may indicate that signals or receptors are involved in forward transport (Lodish et al., 1983; Lodish, 1988) .
T158 47653-47906 Epistemic_statement denotes Similarly, the suggestion that newly synthesized viral spike glycoproteins are present in Golgi membranes at a density severalfold greater than in the ER is also consistent with the existence of signal-driven forward transport (Griffiths et al., 1984) .
T159 47907-48011 Epistemic_statement denotes However, both of these observations might also be reconciled with a nonselective mechanism of transport.
T160 48012-48201 Epistemic_statement denotes Given recent evidence that exit from the ER is linked to the folding of newly synthesized proteins, differential folding rates among proteins may indirectly affect their transport kinetics.
T161 48202-48416 Epistemic_statement denotes Moreover, if the intrinsic rate of transport of glycoproteins through the Golgi is slow relative to the rate of ER exit, then one might also expect a higher concentration of certain passenger proteins in the Golgi.
T162 48541-48647 Epistemic_statement denotes Whether transport through the Golgi complex itself is selective or nonselective is still an open question.
T163 48648-48740 Epistemic_statement denotes After years of descriptive work, the Golgi complex is slowly starting to reveal its secrets.
T164 48741-48937 Epistemic_statement denotes We have now entered an exciting period of research, during which it will become possible to define the molecular mechanisms responsible for generating and maintaining Golgi structure and function.
T165 48938-49268 Epistemic_statement denotes The first phase is already well under way and has been characterized by a search for essential bits and pieces of the Golgi machinery, a number of which have already been found (NSFlsecl8, aSNAPlsecl7, (J-COP, ARF[Stearnsetal., 19901, rab6p[Goudetal., 1990] ,sec7p [Achstetter et al., 19881, and secl4p [Bankaitis et al., 199Oj) .
T166 49269-49394 Epistemic_statement denotes As we have seen, however, it is at present difficult to know precisely what steps are controlled by each of these components.
T167 49671-49740 Epistemic_statement denotes The next phase will have to deal with the questions that have arisen.
T168 49741-49779 Epistemic_statement denotes How many Golgi compartments are there?
T169 49847-49902 Epistemic_statement denotes If so, how do they function and how are they regulated?
T170 49973-50001 Epistemic_statement denotes What is the role of tubules?
T171 50002-50110 Epistemic_statement denotes How does the machinery responsible for forward traffic relate to the machinery controlling homotypic fusion?
T172 50111-50172 Epistemic_statement denotes How is specificity of forward and backward traffic regulated?
T173 50173-50234 Epistemic_statement denotes How does lipid composition and organization affect transport?
T174 50280-50333 Epistemic_statement denotes How do microtubules interact with the Golgi elements?
T175 50334-50464 Epistemic_statement denotes The challenge will be to integrate the information we are now collecting in the context of how the Golgi complex works as a whole.