Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis -SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444–448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis -Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis -Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.     

The overexpression of GMAP-210 blocks anterograde and retrograde transport between the ER and the Golgi apparatus

Golgi Microtubule-Associated Protein (GMAP)-210 is a peripheral coiled-coil protein associated with the cis -Golgi network that interacts with microtubule minus ends. GMAP-210 overexpression has previously been shown to perturb the microtubule network and to induce a dramatic enlargement and fragmentation of the Golgi apparatus (Infante C, Ramos-Morales F, Fedriani C, Bornens M, Rios RM. J Cell Biol 1999; 145: 83–98). We now report that overexpressing GMAP-210 blocks the anterograde transport of both a soluble form of alkaline phosphatase and the hemagglutinin protein of influenza virus, an integral membrane protein, between the endoplasmic reticulum and the cis /medial (mannosidase II-positive) Golgi compartment. Retrograde transport of the Shiga toxin B-subunit is also blocked between the Golgi apparatus and the endoplasmic reticulum. As a consequence, the B-subunit accumulates in compartments positive for GMAP-210. Ultrastructural analysis revealed that, under these conditions, the Golgi complex is totally disassembled and Golgi proteins as well as proteins of the intermediate compartment are found in vesicle clusters distributed throughout the cell. The role of GMAP-210 on membrane processes at the interface between the endoplasmic reticulum and the Golgi apparatus is discussed in the light of the property of this protein to bind CGN membranes and microtubules.     

Exit of nonglycosylated secretory proteins from the rough endoplasmic reticulum is asynchronous in the exocrine pancreas

The path and synchrony of intracellular transport of 12 secretory proteins of the guinea pig exocrine pancreas have been studied in pulse-chase amino acid labeling experiments by quantitative analysis of the individual proteins recovered in subcellular fractions and extracellular samples. Protein fractionation was accomplished by two-dimensional isoelectric focusing/SDS-gel electrophoresis. Use of a double-label protocol allowed correction of the data on a protein-by-protein basis for leakage and adsorption artifacts which accompany tissue homogenization. All the labeled secretory (pro)enzymes, including their isoenzymic forms, were recovered in rough microsomal, Golgi-enriched and granule fractions during their transport to the cell surface. However, major asynchrony was observed at four levels: exit from the rough endoplasmic reticulum; transit through the Golgi complex; entry into granules; and discharge from the cell. Rapid transport rates were observed for trypsinogen, chymotrypsinogen 2, procarboxypeptidase A2, and lipase 2. Slow transport rates were observed for amylase and procarboxypeptidase B. In the presence of carbamylcholine or cholecystokinin stimulation, the times required for 40% discharge of labeled chymotrypsinogen 2, trypsinogen, amylase, and procarboxypeptidase B were 98, 102, 148, and 180 min, respectively. Transport rates did not correlate with isoelectric point, molecular weight, or the presence of carbohydrate. These data suggest that interactions occur within the rough endoplasmic reticulum, either between secretory (nonglyco)-proteins themselves or between such proteins and the cisternal face of the rough endoplasmic reticulum.     

ER-to-Golgi transport: COP I and COP II function (Review)

COP I and COP II coat proteins direct protein and membrane trafficking in between early compartments of the secretory pathway in eukaryotic cells. These coat proteins perform the dual, essential tasks of selecting appropriate cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles. COP II proteins are required for selective export of newly synthesized proteins from the endoplasmic reticulum (ER). COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis-Golgi complex to the ER. Additionally, COP I coat proteins have complex functions in intra-Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi complex.     

Spatial dissection of the cis- and trans-Golgi compartments in the malaria parasite Plasmodium falciparum

The Golgi apparatus forms the heart of the secretory pathway in eukaryotic cells where proteins are modified, processed and sorted. The transport of proteins from the endoplasmic reticulum (ER) to the cis- side of the Golgi complex takes place at specialized ER sub-domains known as transitional ER (tER). We used the Plasmodium falciparum orthologue of Sec13p to analyse tER organization. We show that the distribution of Pf Sec13p is restricted to defined areas of the ER membrane. These foci are juxtaposed to the Golgi apparatus and might represent tER sites. To further analyse cis - to trans -Golgi architecture, we generated a double transfectant parasite line that expresses the Golgi marker Golgi reassembly stacking protein (GRASP) as a green fluorescent protein fusion and the trans- Golgi marker Rab6 as a DsRed fusion protein. Our data demonstrate that Golgi multiplication is closely linked to tER multiplication, and that parasite maturation is accompanied by the spatial separation of the cis- and trans- face of this organelle.     

Coupled induction of exocrine proteins and intracellular compartments involved in the secretory pathway in AR4-2J cells by glucocorticoids

Treatment of AR4-2J cells with dexamethasone at 10 nM for 96 h inhibited cell replication by 75% and increased cell size (30%), protein content (1.6-fold) and protein synthesis (2-fold). The increase in protein synthesis was largely due to a 5 to 10-fold increase in the synthesis of secretory proteins. Amylase activity increased 20 to 30-fold in cellular homogenates and 10 to 20-fold in culture medium. Both in the presence and absence of dexamethasone AR4-2J cells release their secretory proteins by constitutive secretion. The proportion of newly synthesized amylase retained by the cells over the 14 h labeling period increased from 15 to 30% with hormone treatment. As judged by comigration on polyacrylamide gels and Western blots analyzed by immunospecific sera, AR4-2J cells synthesize and secrete the majority of known pancreatic secretory proteins. Dexamethasone increased the synthesis of trypsinogen 12 to 16-fold, chymotrypsinogen 4.5 to 6-fold, the group of procarboxypeptidases 6-fold, and amylase 7 to 10-fold. Messenger RNA levels for trypsinogen, amylase and lipase were each increased 4 to 5-fold. At the ultrastructural level dexamethasone led to significant increases in rough endoplasmic reticulum (RER) (30-fold) and Golgi elements (1.5-fold) and to the de novo appearance of electron-opaque granules (0.1-0.5 microns) which were shown to contain amylase by immunolocalization techniques employing protein A-gold. Dexamethasone also led to the formation of gap junctions between AR4-2J cells. These findings indicate that AR4-2J cells provide a model for differentiation of pancreatic acinar cells which should also be studied for the differentiation markers for the regulated secretory pathway.     

Protein sorting upon exit from the endoplasmic reticulum

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.     

Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER

Background  

In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails.     

Generation of MHC class I peptide antigens by protein processing in the secretory route by furin

Cytosolic degradation of endogenously synthesized proteins by the proteasome and translocation of processed peptides to the endoplasmic reticulum by the transporters associated with antigen presentation constitutes the classical route for antigen presentation by MHC class I proteins. We have previously defined an alternative pathway in the secretory route involving proteolytic maturation of precursor proproteins for chimeric hepatitis B virus secretory core protein HBe containing a class I epitope at its carboxy-terminus. We extend those results by demonstrating that intracellular delivery of the trans -Golgi network protease furin increases both proteolytic maturation and antigen presentation of the chimeric HBe proteins. An additional class I epitope from the HIV envelope gp160 protein was inserted into this COOH-terminal region of two different chimeric HBe proteins. This epitope was also presented to CTL in a transporter-independent manner involving furin, and protein maturation and antigen presentation were also enhanced by furin over-expression. Presentation of this second epitope was restricted by a different class I allele, thus suggesting that antigen presentation by this new pathway may apply to any antigenic epitope and class I molecule. These results define the furin proteolytic maturation pathway of HBe in the secretory route as a general antigen processing route for MHC class I presentation.     

Rer1, a putative transmembrane domain receptor responsible for targeting proteins to the endoplasmic reticulum

Summary Localization of resident proteins provides identity to subcellular compartments. Most proteins depend on a combination of both retention and retrieval to maintain their steady-state distribution. Rerl is a putative receptor protein mediating retrieval of membrane proteins of the endoplasmic reticulum. This retrieval relies on an unusual hydrophobic target sequence, the transmembrane domain. Apart from Rerl, coatomer is also required to retrieve escaped membrane proteins from the early Golgi region back to the endoplasmic reticulum. Current evidence suggests that the Rerl-mediated retrieval of membrane proteins is a general sorting pathway in eukaryotic cells contributing to the maintenance of compartmental identity in the early secretory pathway.     

Studies on pancreatic acinar cells in tissue culture: basal lamina (basement membrane matrix promotes three-dimensional reorganization

Rat pancreatic acinar cells have been dissociated and maintained in culture under specific conditions which allow the retention of their differentiated state and three-dimensional organization. When cultured on a basal lamina (basement membrane) matrix, the cells first formed large monolayer patches and then reorganized themselves into acini-like structures. The cells regained their polarity around luminal spaces which appeared to be sealed off by well developed junctional complexes. Typical microvilli appeared at the "apical" plasma membrane projecting themselves into the luminal spaces. The intracellular organization resembled that of the cells in situ: a well developed rough endoplasmic reticulum located towards the "base" of the cell around a nucleus; a supranuclearly positioned Golgi apparatus and numerous secretory granules located in the "apical" region of the cell. Immunocytochemistry has revealed the presence of two pancreatic enzymes, amylase and chymotrypsinogen, in the various cellular compartments involved in secretion; the rough endoplasmic reticulum and Golgi cisternae as well as in the secretory granules. Biochemical evaluations have also shown the presence of amylase in the acinar cells and culture medium. These results thus demonstrate that dissociated pancreatic acinar cells maintained in culture under specific conditions reaggregate themselves into acini-like structures and retain their differentiated morphology as well as their ability to secrete.     

Protein quality control in the early secretory pathway

Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)-Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. 'Public' and 'private' chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion.     

The efficiency of protein compartmentalization into the secretory pathway

Numerous proteins targeted for the secretory pathway are increasingly implicated in functional or pathological roles at alternative cellular destinations. The parameters that allow secretory or membrane proteins to reside in intracellular locales outside the secretory pathway remain largely unexplored. In this study, we have used an extremely sensitive and quantitative assay to measure the in vivo efficiency of signal sequence-mediated protein segregation into the secretory pathway. Our findings reveal that segregation efficiency varies tremendously among signals, ranging from >95 to <60%. The nonsegregated fraction is generated by a combination of mechanisms that includes inefficient signal-mediated translocation into the endoplasmic reticulum and leaky ribosomal scanning. The segregation efficiency of some, but not other signal sequences, could be influenced in cis by residues in the mature domain or in trans by yet unidentified cellular factors. These findings imply that protein compartmentalization can be modulated in a substrate-specific manner to generate biologically significant quantities of cytosolically available secretory and membrane proteins.     

Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre-Golgi intermediates and Golgi compartments, wild-type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild-type CFTR is present in pre-Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2-fold lower than that found in the endoplasmic reticulum (ER). delta F508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre-Golgi intermediates and peripheral cis Golgi elements, but not in post-Golgi compartments. Our results suggest that the low level of wild-type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in delta F508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.     

Protein retention and localization in the endoplasmic reticulum and the golgi apparatus.

Protein transport along the secretory pathway is supported by a noria of vesicles that bud and fuse, load and unload their cargo from one compartment into the other. However, despite this constant flow-through of proteins and lipids the various compartments of the secretory pathway are able to maintain their own specific composition. Here, we discuss recent insights into mechanisms of protein retention and localization that are necessary for the maintenance of endoplasmic reticulum (ER)- and Golgi-associated typical functions such as protein folding and glycosylation in plant cells.     

Morphometrical and immunocytochemical studies on rat pancreatic acinar cells under control and experimental conditions

Process of amylase and chymotrypsinogen secretion by acinar cells has been studied applying morphological and biochemical approaches. Three conditions were investigated; resting (fed control), cholinergic stimulation and fasting. Morphometrical evaluations have shown that under stimulation, the volume density of zymogen granules decreases drastically while that of the Golgi apparatus increases. This may result from the enhancement in protein processing and the rapid discharge. Quantitation of amylase and chymotrypsinogen immunolabelings present over the cellular compartments has shown that there is no difference in the intensities between tissues from control and stimulated animals. These results imply that total amounts of protein processed by the Golgi apparatus are markedly enhanced primarily because of the increase in size of the organelle, the amounts of protein processed per unit surface remaining unchanged. Under starvation where reduction of secretion occurs, there is a significant decrease in the volume density of the Golgi apparatus but no variation in that of the zymogen granules. However, the morphological aspect of these was markedly altered since many of them present an electron luscent periphery which was devoid of immunolabeling for amylase and chymotrypsinogen. Quantitation of amylase and chymotrypsinogen immunolabelings has shown significant diminution for both enzymes. In both experimental conditions, the volume density of lysosomes was enhanced, however in none of these conditions evidence of crinophagy was observed. The morphometrical and immunocytochemical results were consistent with those obtained from biochemical determination of amylase and chymotrypsinogen contents in tissues. Correlations between results obtained from morphometric and immunocytochemical studies were made leading to a better understanding of the cellular secretory activity during experimental conditions.     

Secretory pathway quality control operating in Golgi,plasmalemmal, and endosomal systems

Exportable proteins that have significant defects in nascent polypeptide folding or subunit assembly are frequently retained in the endoplasmic reticulum and subject to endoplasmic reticulum-associated degradation by the ubiquitin-proteasome system. In addition to this, however, there is growing evidence for post-endoplasmic reticulum quality control mechanisms in which mutant or non-native exportable proteins may undergo anterograde transport to the Golgi complex and post-Golgi compartments before intracellular disposal. In some instances, these proteins may undergo retrograde transport back to the endoplasmic reticulum with re-targeting to the endoplasmic reticulum-associated degradation pathway; in other typical cases, they are targeted into the endosomal system for degradation by vacuolar/lysosomal proteases. Such quality control targeting is likely to involve recognition of features more commonly expressed in mutant proteins, but may also be expressed by wild-type proteins, especially in cells with perturbation of local environments that are essential for normal protein trafficking and stability in the secretory pathway and at the cell surface .     

Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles.

We have determined the concentrations of the secretory proteins amylase and chymotrypsinogen and the membrane proteins KDELr and rBet1 in COPII- and COPI-coated pre-Golgi compartments of pancreatic cells by quantitative immunoelectron microscopy. COPII was confined to ER membrane buds and adjacent vesicles. COPI occurred on vesicular tubular clusters (VTCs), Golgi cisternae, the trans-Golgi network, and immature secretory granules. Both secretory proteins exhibited a first, significant concentration step in noncoated segments of VTC tubules and were excluded from COPI-coated tips. By contrast, KDELr and rBet1 showed a first, significant concentration in COPII-coated ER buds and vesicles and were prominently present in COPI-coated tips of VTC tubules. These data suggest an important role of VTCs in soluble cargo concentration by exclusion from COPI-coated domains.     

A cure for traffic jams: small molecule chaperones in the endoplasmic reticulum

Folding in the endoplasmic reticulum is the limiting step for the biogenesis of most secretory pathway cargo proteins; proteins which fail to fold are initially retained in the endoplasmic reticulum and subsequently often degraded. Mutations that affect secretory protein folding have profound phenotypes irrespective of their direct impact on protein function, because they prevent secretory proteins from reaching their final destination. When unicellular organisms are stressed by fluctuation of temperature or ionic strength, they synthesize high concentrations of small molecules such as trehalose or glycerol to prevent protein denaturation. These osmolytes can also stabilize mutant secretory proteins and allow them to pass secretory protein quality control in the endoplasmic reticulum. Specific ligands and cofactors such as ions, sugars, or peptides have similar effects on specific defective proteins and are beginning to be used as therapeutic agents for protein trafficking diseases.     

Concentration of amylase along its secretory pathway in the pancreatic acinar cell as revealed by high resolution immunocytochemistry

Summary The modified protein A-gold immunocytochemical technique was applied to the localization of amylase in rat pancreatic acinar cells. Due to the good ultrastructural preservation of the cellular organelles obtained on glutaraldehyde-fixed, osmium tetroxide-postfixed tissue, the labelling was detected with high resolution over the cisternae of the rough endoplasmic reticulum (RER), the Golgi apparatus, the condensing vacuoles, the immature pre-zymogen granules, and the mature zymogen granules. Over the Golgi area, the labelling was present over the transitional elements of the endoplasmic reticulum, some of the smooth vesicular structures at thecis- andtrans-faces and all the different Golgi cisternae. The acid phosphatase-positive rigidtrans-cisternae as well as the coated vesicles were either negative or weakly labelled. Quantitative evaluations of the degree of labelling demonstrated an increasing intensity which progresses from the RER, through the Golgi, to the zymogen granules and have identified the sites where protein concentration occurs. The results obtained have thus demonstrated that amylase is processed through the conventional RER-Golgi-granule secretory pathway in the pancreatic acinar cells. In addition a concomitance has been found between some sites where protein concentration occurs: thetrans-most Golgi cisternae, the condensing vacuoles, the pre- and the mature zymogen granules, and the presence of actin at the level of the limiting membranes of these same organelles as reported previously (Bendayan, 1983). This suggests that beside their possible role in transport and release of secretory products, contractile proteins may also be involved in the process of protein concentration.