Brefeldin A-induced alterations in processing of MHC class II-Ii complex depend upon microtubular function

The role of microtubules in the brefeldin A (BFA)-associated relocation of major histocompatibility complex (MHC) class II αβ chains (αβ) and the invariant chain (Ii) was characterized in Raji cells by the use of nocodazole (ND). BFA blocked the transport of αβIi proteins through the Golgi and redistributed them to the endoplasmic reticulum (ER) along with Golgi-resident enzymes. The result of the colocalization of processing enzymes and newly synthesized proteins was a downshift of αβIi molecular weight (MW) of 2 kDa, and their resistance to endoglycosidase H (endo H) after 6 hr of chase. ND by itself had no effect on the processing and transport of αβ to the cell surface. The addition of ND to BFA-treated cells downshifted αβIi by 4 kDa. Additionally, αβIi proteins remained sensitive to neuraminidase after 16 hr of chase. In vitro α-mannosidase treatment of immunoprecipitated αβIi generated a similar 4-kDa downshift of MW. Either 1-deoxymannojirimycin (DJN) or swainsonine (SWN) blocked the MW downshift caused by BFA + ND treatment. These observations indicated that in Raji cells, most of the BFA-associated relocations of cis-, medial Golgi proteins, and the addition of sialic acid from the trans-Golgi were microtubule-independent. The retrograde transport of the medial Golgi enzyme N-acetylglucosamine transferase, however, required microtubular function. Microtubule disrupters could affect BFA treatment of viral infections by further disrupting viral protein processing. Am. J. Hematol. 54: 282–287, 1997. © 1997 Wiley-Liss, Inc.     

Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells.

The effect of temperature on secretory protein transport was studied by cell fractionation of rat pancreatic lobules, pulse-labeled in vitro with [35S]methionine and chased for 60 min at 16, 20, or 37 degrees C. Chase at 37 degrees C allowed secretory proteins to reach a zymogen granule fraction, whereas chase at 16 or 20 degrees C led to their extensive retention in a total microsomal fraction. To pinpoint the sites of transport inhibition, total microsomes were subfractionated by flotation in a sucrose density gradient. Five bands were resolved, of which the heaviest or B1 (density = 1.20 g/ml) consisted primarily of rough microsomes. The lighter fractions, B2 (1.17 g/ml), B3 (1.15 g/ml), and B4 (1.14-1.13 g/ml), consisted primarily of smooth vesicles derived from Golgi elements. B4 had the highest specific activity for galactosyltransferase, a trans Golgi cisternal marker; B2, B3, and B4 are assumed to represent cis, middle, and trans Golgi subcompartments, respectively. At the end of a 2-min pulse, a single peak of labeled proteins colocalized with B1. During subsequent 60-min chases, labeled proteins advanced to B2 at 16 degrees C and to B3 at 20 degrees C. At 37 degrees C the radioactivity remaining in the total microsomal fraction was distributed among four peaks (B1-B4). The results indicate that transport from the endoplasmic reticulum to the Golgi complex is strongly inhibited below 20 degrees C. At 16 degrees C, the bulk of the cohort of labeled secretory proteins is still in the rough endoplasmic reticulum, but its advancing front reaches cis Golgi elements. At 20 degrees C, the front advances to a middle Golgi compartment, and at 37 degrees C most of the cohort (approximately 70%) reaches condensing vacuoles and zymogen granules. It is concluded that transport steps along the endoplasmic reticulum-plasmalemma pathway have distinct temperature requirements.     

Mammalian Erv46 localizes to the endoplasmic reticulum-Golgi intermediate compartment and to cis-Golgi cisternae

Yeast endoplasmic reticulum (ER) vesicle protein Erv46p is a novel membrane protein involved in transport through the early secretory pathway. Investigation of mammalian Erv46 (mErv46) reveals that it is broadly expressed in tissues and protein-secreting cells. By immunofluorescence microscopy, mErv46 displays a crescent-shaped perinuclear staining pattern that is characteristic of the Golgi complex. Quantitative immunoelectron microscopy indicates that mErv46 is restricted to the cis face of the Golgi apparatus and to vesicular tubular structures between the transitional ER and cis-Golgi. Minor amounts of mErv46 reside in ER membranes and later Golgi cisternae. On Brefeldin A treatment, mErv46 redistributes to punctate structures that costain for ERGIC53. Depletion of mErv46 protein by RNA interference caused no apparent structural changes in the intermediate compartment or Golgi complex. These findings place mErv46 in a group of itinerant proteins that cycle between the ER and Golgi compartments such as ERGIC53 and the p24 proteins.     

The reorganization of the Golgi complex in anoxic pancreatic acinar cells

An extensive reorganization of the Golgi complex (GC) was found in the acinar cells of pancreatic lobules incubated in vitro under conditions which inhibited ATP synthesis and thereby blocked the intracellular transport of secretory proteins. After 15-min incubation under N2 or in the presence of 1 mM dinitrophenol (DNP), transitional elements of the endoplasmic reticulum (ER) lost their protrusions, small peripheral Golgi vesicles decreased drastically in number, and fibrillar aggregates approximately 0.2 to 0.5 micron in diameter appeared on the cis side of the stacks of Golgi cisternae. These aggregates often contained vesicle-free, small (approximately 40 nm), globular cages and, occasionally, vesicle-free, clathrin-like cages. Fibrillar aggregates were also observed on the trans side of Golgi stacks. Other changes included the proliferation of GERL-elements ("rigid lamellae") and a striking increase in the population of coated vesicles trans to the Golgi complex and throughout the apical region of the anoxic acinar cells. All changes were found to be reversible provided the cells were incubated for less than 1 h under N2. These observations suggest that the fibrillar elements and the associated cages described in this study may play a role in the vesicular transport of newly synthesized proteins from the ER to the Golgi complex. Further work is needed to explore this possibility.     

Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm.

The yeast Sec23 protein is required in vivo and in vitro for transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Ultrastructural localization of the Sec23p mammalian homologue (detected by antibody cross-reaction) in exocrine and endocrine pancreatic cells shows a specific distribution to the cytoplasmic zone between the transitional ER cisternae and Golgi apparatus where it appears associated with the tubular protuberances of the transitional ER cisternae, as well as with a population of vesicles, and surrounding cytoplasm. When ER-Golgi transport is interrupted with an energy poison, protuberances and transfer vesicles markedly decrease but Sec23p immunoreactive sites remain in the transitional cytoplasm not apparently tethered by membrane attachment. This unanticipated degree of organization suggests that cytosolic proteins, such as Sec23p, may be retained in specialized areas of the cytoplasm. A structure within the transitional zone may organize the flux of transport vesicles and Sec proteins so as to ensure efficient protein traffic in this limb of the secretory pathway.     

Blockade by brefeldin A of intracellular transport of secretory proteins in mouse pituitary cells: effects on the biosynthesis of thyrotropin and free alpha-subunits

We examined the effect of brefeldin A (BFA), a drug that inhibits the intracellular translocation of newly synthesized glycoproteins, on the biosynthesis of TSH and free alpha-subunits by pituitary tissue from hypothyroid mice. Incubation of tissue with 5 or 10 micrograms BFA/ml for 3.5 h caused marked dilatation of rough endoplasmic reticulum (RER) and mild swelling of Golgi in all pituitary cell types. As judged by incorporation of [35S]Met into acid-insoluble radioactivity, BFA at a concentration of 5 micrograms/ml did not substantially inhibit protein synthesis, but markedly reduced protein secretion. After a 2-h pulse with [35S]Met, followed by a 4-h chase, BFA at 5 micrograms/ml reduced the release of TSH and free alpha-subunits into the medium by 94% and 99%, respectively; subunits that accumulated within cells were forms with mol wt 2000-4000 less than normal. BFA also partially inhibited the release into the medium of TSH or free alpha-subunits labeled with [3H]fucose or [35S]SO4, but this effect was less marked than that for [35S]Met-labeled subunits. Both the morphological and the isotopic data suggest that BFA blocks transport of secretory proteins between rough endoplasmic reticulum and Golgi of pituitary cells, although transport within the Golgi may also be affected to some extent.     

A C-terminally-anchored Golgi protein is inserted into the endoplasmic reticulum and then transported to the Golgi apparatus.

Unlike conventional membrane proteins of the secretory pathway, proteins anchored to the cytoplasmic surface of membranes by hydrophobic sequences near their C termini follow a posttranslational, signal recognition particle-independent insertion pathway. Many such C-terminally-anchored proteins have restricted intracellular locations, but it is not known whether these proteins are targeted directly to the membranes in which they will ultimately reside. Here we have analyzed the intracellular sorting of the Golgi protein giantin, which consists of a rod-shaped 376-kDa cytoplasmic domain followed by a hydrophobic C-terminal anchor sequence. Unexpectedly, we find that giantin behaves like a conventional secretory protein in that it inserts into the endoplasmic reticulum (ER) and then is transported to the Golgi. A deletion mutant lacking a portion of the cytoplasmic domain adjacent to the membrane anchor still inserts into the ER but fails to reach the Golgi, even though this mutant has a stable folded structure. These findings suggest that the localization of a C-terminally-anchored Golgi protein involves at least three steps: insertion into the ER membrane, controlled incorporation into transport vesicles, and retention within the Golgi.     

Redistribution of clathrin heavy and light chains in anoxic pancreatic acinar cells

In the present study, antibodies directed against clathrin light (33 kDa-36 kDa) and heavy (180 kDa) chains were used to confirm, by immunocytochemistry, that coated vesicles increase in number in the exocrine cells of pancreatic lobules incubated under anoxic (N2) conditions. The same antibodies were used to check whether or not the fibrillar aggregates, which appear under the same conditions on the cis side of the Golgi stacks of these cells, contain clathrins. By immunofluorescence, clathrin light chains were localized among zymogen granules and in small masses at the periphery of the Golgi complex in acinar cells incubated under N2. By electron microscopy, antibodies for light and heavy chains reacted with numerous coated vesicles located in clusters on the trans side of the Golgi stacks, scattered individually or in small clusters among zymogen granules throughout the apical region of the cell, and associated with both the apical and basolateral plasmalemma of the exocrine cells incubated under N2. The fibrillar aggregates cis to the Golgi complex stained less intensely and much less uniformly than the coats of the coated vesicle population. These findings suggest that the fibrillar aggregates which characteristically appear on the cis side of the Golgi stacks under anoxic conditions contain only small amounts of focally distributed clathrins. Their main components are probably other proteins whose relationship (if any) to clathrins and other clathrin cage constituents remains to be investigated. The findings also indicate that pancreatic acinar cells redistribute large amounts of clathrin, presumably from preexisting pools, when the transport of secretory and other proteins into the Golgi complex is inhibited. In control cells (incubated under O2-CO2), clathrin antigens had the same structural associations as in anoxic cells but the reactive sites were considerably less numerous and the reaction less intense.     

Structural domains and molecular lifestyles of insulin and its precursors in the pancreatic Beta cell

Summary Insulin is both produced and degraded within the pancreatic Beta cell. Production involves the synthesis of the initial insulin precursor preproinsulin, which is converted to proinsulin shortly after (or during) translocation into the lumen of the rough endoplasmic reticulum. Proinsulin is then transported to the trans-cisternae of the Golgi complex where it is directed towards nascent secretory granules. Conversion of proinsulin to insulin and C-peptide arises within secretory granules, and is dependent upon their acidification. Granule contents are discharged by exocytosis in response to an appropriate stimulus. This represents the regulated secretory pathway to which more than 99% of proinsulin is directed in Beta cells of a healthy individual. An alternative route also exists in the Beta cell, the constitutive secretory pathway. It involves the rapid transfer of products from the Golgi complex to the plasma membrane for immediate release, with, it is supposed, little occasion for prohormone conversion. Even if delivered appropriately to secretory granules, not all insulin is released; some is degraded by fusion of granules with lysosomes (crinophagy). Each event in the molecular lifestyles of insulin and its precursors in the Beta cell will be seen to be governed by their own discrete functional domains. The identification and characterisation of these protein domains will help elucidate the steps responsible for delivery of proinsulin to secretory granules and conversion to insulin. Understanding the molecular mechanism of these steps may, in turn, help to explain defective insulin production in certain disease states including diabetes mellitus.Presented in part as the 25th Minkowski Prize Lecture at the EASD Annual Meeting. Copenhagen, September, 1990     

Biosynthesis and sorting of myeloperoxidase in hematopoietic cells

The neutrophil granulocytes have a critical role in innate immunity through killing of phagocytized microorganisms, in which myeloperoxidase (MPO) participates. MPO is stored in cytoplasmic azurophil lysosome-like granules together with other antibiotic proteins and digestive enzymes. During passage in the secretory pathway pro-MPO is folded, subjected to oligosaccharide modification, and retrieval from constitutive secretion to become targeted to azurophil granules for final processing and storage. Propeptide-deleted MPO precursor was found not to be processed to mature MPO and not to be targeted for storage but instead degraded or secreted. This indicated that the propeptide of the MPO precursor was a prerequisite for the final processing and granule targeting of proMPO. When the MPO propeptide was expressed as a chimera with a normally secretory protein, the ER retention of the chimera was prolonged compared with that of the native protein. Thus, the propeptide of MPO precursor may also mediate the normally long ER-residence of proMPO. Both mature MPO and secreted proMPO contained complex oligosaccharide side chains indicating that proMPO and, thus, mature MPO has passed the medial Golgi stack where complex oligosaccharides are formed, and exited at TGN like other proteins targeted for azurophil granules.     

Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum

Mnn9p is a component of two distinct multiprotein complexes in the Saccharomyces cerevisiae cis-Golgi that have both been shown to have alpha-1,6-mannosyltransferase activity in vitro. In one of these complexes, Mnn9p associates with four other membrane proteins, Anp1p, Mnn10p, Mnn11p, and Hoc1p, whereas the other complex consists of Mnn9p and Van1p. Members of the Mnn9p-containing complexes were incorporated into COPII vesicles made in vitro from endoplasmic reticulum (ER) membranes isolated from cycloheximide-treated cells. This behavior is consistent with an active Golgi to ER recycling process. To examine this path in vivo, we monitored retrograde transport of subunits of the complex in cells blocked in anterograde transport from the ER. In this situation, specific relocation of the proteins from the Golgi to the ER was observed in the absence of new protein synthesis. Conversely, when retrograde transport was blocked in vivo, subunits of the mannosyltransferase complex accumulated in the vacuole. Packaging of Mnn9p in COPI-coated vesicles from purified Golgi membranes was also investigated using a coatomer-dependent vesicle budding assay. Gradient fractionation experiments showed that Mnn9p and the retrograde v-SNARE, Sec22p, were incorporated into COPI-coated vesicles. These observations indicate that the Mnn9p-containing mannosyltransferase complexes cycle back and forth between the ER and Golgi.     

Cytochemical study of secretory process in transplantable insulinoma of syrian golden hamster.

Electron microscopy, including phosphatase cytochemistry, indicates that the secretory granules of an insulinoma producing proinsulin and insulin are packaged by the endoplasmic reticulum (ER) and especially by a specialized region of ER which we call GERL because of the spatial relationship of this region to the Golgi apparatus and its apparent role in producing lysosomes. The granules are not derived from the Golgi apparatus. Preliminary evidence suggests this may be true also of pancreatic beta-cells.     

Phospholipase D is present on Golgi-enriched membranes and its activation by ADP ribosylation factor is sensitive to brefeldin A.

ADP ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that regulates the binding of coat proteins to membranes and is required for several stages of vesicular transport. ARF also stimulates phospholipase D (PLD) activity, which can alter the lipid content of membranes by conversion of phospholipids into phosphatidic acid. Abundant PLD activity was found in Golgi-enriched membranes from several cell lines. Golgi PLD activity was greatly stimulated by ARF and GTP analogs and this stimulation could be inhibited by brefeldin A (BFA), a drug that blocks binding of ARF to Golgi membranes. Furthermore, in Golgi membranes from BFA-resistant PtK1 cells, basal PLD activity was high and not stimulated by exogenous ARF or GTP analogs. Thus, ARF activates PLD on the Golgi complex, suggesting a possible link between transport events and the underlying architecture of the lipid bilayer.     

Concentrating hormones into secretory granules: layers of control

Soluble protein hormones are concentrated and stored in secretory granules The cisternal maturation model for transport of proteins through the Golgi complex allows a major role for formation of reversible aggregates as a means of both concentrating and sorting hormones, since soluble proteins will be removed in small vesicles, leaving behind the aggregated hormones. The storage of secretory granule proteins, however, is more selective than would be expected if passive aggregation were the only process involved. Aggregation of hormones in the secretory pathway may not be completely passive, but may be controlled by the cells. In addition to aggregation, other layers of sorting must exist, because there is selective retention of proteins after aggregation or packaging into granules.     

The asynchronous transport of secretory proteins in the exocrine pancreas. Compatibility with the hypothesis of a paragranular pathway?

An asynchronous transport of individual secretory proteins has been recently described in the pancreas. This asynchrony was observed in both unstimulated and stimulated conditions. It has also been proposed that unstimulated and stimulated secretions correspond to distinct secretory processes. Indeed according to that hypothesis, under resting conditions, a small fraction of the newly synthesized secretory proteins are channeled into a paragranular (vesicle) pathway while the residual proteins are packaged in the zymogen granules. These zymogen granules eventually move to the cell surface where their content is extruded by exocytosis. Under stimulated conditions the latter process is accelerated. Since the same type of asynchrony is observed under resting and stimulated conditions in the pancreatic juice, one can wonder if the hypothesis of a paragranular pathway is compatible with the observed asynchrony. In this review, an explanation is presented to account for the facts that following pulse and chase labelling, two waves of labelled proteins are released under resting secretions and secondly that asynchrony is maintained in both resting and stimulated conditions.     

Mechanism of acute pancreatitis Cellular and subcellular events

A membrane-bound system through which secretory and lysosomal proteins travel in a vectorial fashion is essential for the preserved integrity of pancreatic acinar cells. This system is composed of an ordered array of compartments, such as the rough endoplasmic reticulum, the Golgi complex, lysosomes, and secretory granules. As a principle, in acute pancreatitis the final steps of this transport seem to be disturbed. Caerulein-induced pancreatitis is a valuable experimental model for studying altered intracellular transport, and compartmentation of lysosomal and digestive enzymes. The formation of enlarged secretory vacuoles containing lysosomal and digestive enzymes is paralleled by the activation of lysosomes and degradation of cellular organelles in autophagosomes. On the level of secretory and autophagic vacuoles, activation of serine proteases occurs, which in addition to increasing lysosomal enzyme activities can represent the initial stage for acinar cell destruction and the development of pancreatitis.     

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (βIΣ) and ankyrin (Ank_G119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as Ank_G119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.     

Insulin immunoreactive sites demonstrated in the Golgi apparatus of pancreatic B cells.

Insulin immunoreactive sites were localized in the Golgi apparatus of pancreatic B cells by light and electron microscopy. Identification of the Golgi apparatus by immunofluorescence required the prior degranulation of B cells with glibenclamide to reduce the insulin immunostaining due to secretory granules. In such cells, insulin immunofluorescence revealed brightly stained, crescent-shaped strands with form and location super-imposable on that of Golgi complexes seen in thin sections of the same cells. With the electron microscope, the insulin immunoreactive sites revealed by the protein A/gold technique were localized in the cisternae and vesicles of the Golgi apparatus of glibenclamide-treated and control B cells and over maturing and mature secretory granules. The quantitative evaluation of the intensity of the insulin immunoreactive sites in the Golgi apparatus revealed a density of sites 4 times more than cellular background values. The demonstration of insulin immunoreactivity in the Golgi apparatus provides direct evidence for the involvement of this compartment in the transport and maturation of proinsulin into insulin.     

Identification of a gene required for membrane protein retention in the early secretory pathway.

The yeast SEC12 gene product (Sec12p) is an integral membrane protein required for the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Although this protein is almost exclusively localized in the ER, a significant fraction of Sec12p is modified by an enzyme that resides in the early compartment of the Golgi apparatus, suggesting that Sec12p is cycling between the ER and the early Golgi. We have taken a genetic approach to investigate the retention mechanism of Sec12p. Analysis of mutants that are defective in the retention of the Sec12-Mf alpha 1 fusion protein in the early secretory compartments has identified a gene, RER1. A recessive mutation in RER1 causes mislocalization of the authentic Sec12p as well as two different Sec12 fusion proteins to the late Golgi apparatus and even to the cell surface. However, the rer1 mutant is not defective in the retention of an ER-resident soluble protein, BiP, suggesting that soluble and membrane proteins are retained in the ER by distinct mechanisms.