ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments

The oligosaccharide processing intermediates of the vesicular stomatitis virus strain ts045 G protein were used to identify ATP- and temperature-sensitive steps in the constitutive pathway of protein transfer to the cell surface. In addition to the initial ATP-sensitive step required for export from the endoplasmic reticulum (Balch, W. E., Elliott, M. M., and Keller, D. S. (1986) J. Biol. Chem. 261, 14681-14689), two distinct ATP-sensitive steps functionally dissect the Golgi into at least 3 compartments: a cis compartment containing the trimming enzyme mannosidase I, a medial compartment conferring resistance to endoglycosidase H, and a trans compartment containing terminal glycosyl transferases. A fourth ATP-sensitive step is required for export of G protein from the trans Golgi to the cell surface. A high threshold of cellular ATP (70% of the control) was required for maximal rates of transport between Golgi compartments. Transport between compartments is inhibited at 40% of the normal cellular ATP pool. Only a single temperature-sensitive step localized to the endoplasmic reticulum inhibited transport of ts045 G protein to the cell surface. The data suggest that ATP-sensitive steps punctuate transport of protein between compartmental boundaries of the secretory pathway.     

Membrane traffic between secretory compartments is differentially affected during mitosis.

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.     

Phospholipase D-dependent and -independent mechanisms are involved in milk protein secretion in rabbit mammary epithelial cells

Phospholipase D has been implicated in membrane traffic in the secretory pathway of yeast and of some mammalian cell lines. Here we investigated the involvement of phospholipase D in protein transport at various steps of the secretory pathway of mammary epithelial cells. Treatment of rabbit mammary explants with butanol, which blocks the formation of phosphatidic acid, decreased the secretion of caseins and to a lesser extent that of whey acidic protein. Butanol interfered with both the endoplasmic reticulum to Golgi complex transport of the caseins and secretory vesicle formation from the trans-Golgi network. In contrast, the transport of whey acidic protein to the Golgi was less affected. Activation of protein kinase C enhanced the overall secretion of both markers and interestingly, this stimulation of secretion was maintained for whey acidic protein in the presence of butanol. Transphosphatidylation assays demonstrated the existence of a constitutive phospholipase D activity which was stimulated by the activation of protein kinase C. We conclude that phospholipase D plays a role in casein transport from the endoplasmic reticulum to the Golgi and in the secretory vesicle formation from the trans-Golgi network. Moreover, our results suggest a differential requirement for phospholipase D in the secretion of caseins and that of whey acidic protein.     

Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome

The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human alpha1-antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded alpha1-antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded alpha1-antitrypsin for degradation by the proteasome.     

Ca(2+)-independent phospholipase A2 participates in the vesicular transport of milk proteins

Changes in the lipid composition of intracellular membranes are believed to take part in the molecular processes that sustain traffic between organelles of the endocytic and exocytic transport pathways. Here, we investigated the participation of the calcium-independent phospholipase A2 in the secretory pathway of mammary epithelial cells. Treatment with bromoenol lactone, a suicide substrate which interferes with the production of lysophospholipids by the calcium-independent phospholipase A2, resulted in the reduction of milk proteins secretion. The inhibitor slowed down transport of the caseins from the endoplasmic reticulum to the Golgi apparatus and affected the distribution of p58 and p23, indicating that the optimal process of transport of these proteins between the endoplasmic reticulum, the endoplasmic reticulum/Golgi intermediate compartment and/or the cis-side of the Golgi was dependent upon the production of lysolipids. Moreover, bromoenol lactone was found to delay the rate of protein transport from the trans-Golgi network to the plasma membrane. Concomitantly, membrane-bound structures containing casein accumulated in the juxtanuclear Golgi region. We concluded from these results that efficient formation of post-Golgi carriers also requires the phospholipase activity. These data further support the participation of calcium-independent phospholipase A2 in membrane trafficking and shed a new light on the tubulo/vesicular transport of milk protein through the secretory pathway.     

Ca2+-independent phospholipase A2 participates in the vesicular transport of milk proteins

Changes in the lipid composition of intracellular membranes are believed to take part in the molecular processes that sustain traffic between organelles of the endocytic and exocytic transport pathways. Here, we investigated the participation of the calcium-independent phospholipase A2 in the secretory pathway of mammary epithelial cells. Treatment with bromoenol lactone, a suicide substrate which interferes with the production of lysophospholipids by the calcium-independent phospholipase A2, resulted in the reduction of milk proteins secretion. The inhibitor slowed down transport of the caseins from the endoplasmic reticulum to the Golgi apparatus and affected the distribution of p58 and p23, indicating that the optimal process of transport of these proteins between the endoplasmic reticulum, the endoplasmic reticulum/Golgi intermediate compartment and/or the cis-side of the Golgi was dependent upon the production of lysolipids. Moreover, bromoenol lactone was found to delay the rate of protein transport from the trans-Golgi network to the plasma membrane. Concomitantly, membrane-bound structures containing casein accumulated in the juxtanuclear Golgi region. We concluded from these results that efficient formation of post-Golgi carriers also requires the phospholipase activity. These data further support the participation of calcium-independent phospholipase A2 in membrane trafficking and shed a new light on the tubulo/vesicular transport of milk protein through the secretory pathway.     

Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface

Novel subcellular fractionation procedures and pulse-chase techniques were used to study the intracellular transport of the microvillar membrane hydrolases sucrase-isomaltase and dipeptidylpeptidase IV in the differentiated colon adenocarcinoma cell line Caco-2. The overall rate of transport to the cell surface was two fold faster for dipeptidylpeptidase IV than for sucrase-isomaltase, while no significant differences were observed in transport rates from the site of complex glycosylation to the brush border. The delayed arrival of sucrase-isomaltase in the compartment where complex glycosylation occurs was only in part due to exit from the endoplasmic reticulum. A major slow-down could be ascribed to maturation in and transit of this enzyme through the Golgi apparatus. These results suggest that the observed asynchronism is due to more than one rate-limiting step along the rough endoplasmic reticulum to trans-Golgi pathway.     

Reduced extracellular pH reversibly inhibits oligomerization, intracellular transport, and processing of the influenza hemagglutinin in infected Madin-Darby canine kidney cells

Incubation of Madin-Darby canine kidney cells infected with influenza virus in medium of pH 5.8-6.0 blocks transport of newly synthesized hemagglutinin and processing of the hemagglutinin oligosaccharides to a form resistant to endo H digestion. Upon restoration of the culture medium to pH 7.4, arrested hemagglutinin is processed and then appears on the cell surface, indicating that exclusively transport and not oligosaccharide processing is inhibited. Based upon kinetic data and localization of blocked hemagglutinin by immunofluorescence, the point of inhibition appears to be a discrete step in transport located in a pre-Golgi compartment. This conclusion is supported by the observation that trimerization of hemagglutinin, which is believed to occur in the endoplasmic reticulum, is also inhibited by acidic medium.     

Cell-free systems to study vesicular transport along the secretory and endocytic pathways

Proteins bound for the cell surface, lysosomes, and secretory storage granules share a common pathway of intracellular transport. After their synthesis and translocation into the endoplasmic reticulum, these proteins traverse the secretory pathway by a series of vesicular transfers. Similarly, nutrient and signaling molecules enter cells by endocytosis, and move through the endocytic pathway by passage from one membrane-bound compartment to another. Little is known about the mechanisms by which proteins are collected into transport vesicles, or how these vesicles form, identify their targets, and subsequently fuse with their target membranes. An important advance toward our understanding these processes has come from the establishment of cell-free systems that reconstitute vesicular transfers in vitro. It is now possible to measure, in vitro, the transport of proteins from the endoplasmic reticulum to the Golgi, between Golgi cisternae, and the formation of transport vesicles en route from the trans Golgi network to the cell surface. Along the endocytic pathway, cell-free systems are available to study clathrin-coated vesicle formation, early endosome fusion, and the fusion of late endosomes with lysosomes. Moreover, the selective movement of receptors between late endosomes and the trans Golgi network has also been reconstituted. The molecular mechanisms of vesicular transport are now amenable to elucidation.     

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.     

Plasma cell immunoglobulin M molecules. Their biosynthesis, assembly, and intracellular transport

Immunoglobulin M (IgM)-secreting murine plasmablasts have been used to explore the cytologic site(s) of the successive modifications of the polypeptide H and L chains (steps of glycosylation, chain assembly, and polymerization) which occur during intracellular transport (ICT) and the interrelationships between these events. A combination of pulse- chase biosynthetic labeling protocols (using amino acids and sugars), subcellular fractionation, and electron microscope autoradiography was used in conjunction with inhibitors of glycosylation and agents (carboxyl cyanide m-chlorophenyl hydrazone [CCCP] and monensin) which block Ig exit from the rough endoplasmic reticulum (RER) or Golgi cisternae. The data are consistent with the following conclusions: (1) Sugar addition and modification occur in three main steps: (a) en bloc addition of core sugars to nascent H chains, (b) partial trimming of these oligosaccharide chains in the RER, (c) quasiconcerted addition of terminal sugars (galactose, fucose, and sialic acid) in a very distal compartment between monensin-sensitive Golgi cisternae and the cell surface. (2) H and L chain assembly occurs between nascent H chains and a pool of free light chains present in the RER, followed by interchain disulfide bonding and rapid assembly of monomers into J chain- containing pentamers in the RER. Small amounts of various apparently non-obligatory intermediates in polymerization are also formed. (3) Carbohydrate addition is not required for chain assembly, polymerization, and secretion since completely unglycosylated chains (synthesized in the presence of deoxyglucose or tunicamycin) undergo polymerization and are secreted (although at a reduced rate). (4) Surface 8s IgM molecules do not represent a step in the IgM secretory pathway.     

Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.     

Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.     

Free Ricin A Chain Reaches an Early Compartment of the Secretory Pathway before It Enters the Cytosol

During the intoxication of mammalian cells by ricin, the catalytically active A chain must cross the membrane of an intracellular compartment in order to reach its ribosomal substrates in the cytosol. The actual site of ricin A chain translocation is unclear, and conflicting views hold that it enters the cytosol from endosomes or from an early compartment of the secretory pathway, possibly the lumen of the endoplasmic reticulum. Here we show that treating cells with brefeldin A, or transiently overexpressing mutant GTPases known to inhibit biochemical complexes mediating anterograde and retrograde transport between the endoplasmic reticulum and the Golgi complex, protected cells from intoxication by free ricin A chain. These data indicate that ricin A chain, either free or as part of intact ricin, reaches an early compartment of the secretory pathway before translocation into the cytosol occurs.     

Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells.

Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intracellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.     

Oligosaccharide transport: pumping waste from the ER into lysosomes.

N-glycans play important roles during the folding and secretion of glycoproteins. Surprisingly, during the N-glycosylation of glycoproteins, considerable amounts of unconjugated polymannose-type oligosaccharides ('free OS') are generated. Although free oligosaccharides have no known function in mammalian cells, a sophisticated cellular machinery enables them to be cleared from the endoplasmic reticulum (ER) into the cytosol and then re-enter the endomembrane system at the level of the lysosome. One possible function of this pathway is to stop free OS from interfering with the carbohydrate-dependent aspects of glycoprotein folding and transport along the secretory pathway.     

The carboxyl-terminal valine is required for transport of glycoprotein CD8 alpha from the endoplasmic reticulum to the intermediate compartment

There is evidence that a carboxyl-terminal valine residue is an anterograde transport signal for type I transmembrane proteins. Removal of the signal would either delay glycosylation in the Golgi complex of proteins destined to recycle to the endoplasmic reticulum or determine accumulation in the endoplasmic reticulum of newly synthesized proteins destined for the plasma membrane. We used the human CD8 alpha glycoprotein to investigate the role of the carboxyl-terminal valine in the exocytic pathway. Using immunofluorescence light microscopy, metabolic labeling, and cell fractionation, we demonstrate that removal of the carboxyl-terminal valine residue delays transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment. Removal of the residue did not affect the other steps of the exocytic pathway or the folding/dimerization and glycosylation processes. Therefore, it is likely that this signal plays a role in the transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment either before or during the formation of the transport vesicles that drive the exit the protein from the endoplasmic reticulum.     

Effects of hypertonic and sodium-free medium on transport of a membrane glycoprotein along the secretory pathway in cultured mammalian cells

Incubation of cultured cells in hypertonic medium and sodium-free medium have been shown to block transport at two different stages along the endocytic pathway. To determine the effects of these treatments on the exocytic pathway, we studied the transport of the membrane glycoprotein of vesicular stomatitis virus (VSV-G) in cells infected with tsO45 mutant virus. This mutant synthesizes a VSV-G that accumulates in the endoplasmic reticulum (ER) when cells are incubated at 39.5 degrees C. In addition, VSV-G accumulates in the post-ER pre-Golgi compartment when cells are incubated at 15 degrees C and in the trans-Golgi network (TGN) when cells are incubated at 18 degrees C. Upon transfer of cells to 32 degrees C in control medium, VSV-G exits each of these compartments and is transported to the cell surface. Incubation in sodium-free medium at 32 degrees C did not block transport from any of these three compartments. In contrast, incubation in hypertonic medium blocked export from the ER, transport from the pre-Golgi compartment to the Golgi complex, and transport from the TGN to the cell surface. Our results, in combination with previous studies, suggest that hypertonic medium blocks at least five distinct transport steps; the three exocytic steps described here, endocytosis from the cell surface, and transport of cell surface proteins into the Golgi complex. This raises the possibility that vesicular transport in different parts of the cell shares common elements that are inhibited by this treatment.     

Microtubules and protein secretion in rat lacrimal glands: localization of short-term effects of colchicine on the secretory process

The pathway and kinetics of the secretory protein transport in rat lacrimal exorbital gland have been established by an in vitro time- course radioautographic study of pulse-labeled protein secretion. The colchicine-sensitive steps have been localized by using the drug at various times with respect to the pulse labeling of proteins. Colchicine (10 microM) does not block any step of the secretory protein transport, but when introduced before the pulse it decreases the transfer of labeled proteins from the rough endoplasmic reticulum to the Golgi area, suppressing their temporary accumulation in the Golgi area before any alteration of this organelle is detectable. Moreover, colchicine inhibits protein release only from the secretory granules formed in its presence because the peroxidase discharge is diminished 1 h after colchicine addition, and the secretion of newly synthesized proteins is strongly inhibited only when colchicine is introduced before secretory granule formation. Morphometric studies show that there is a great increase of secondary lysosomes, related to crinophagy, as early as 40-50 min after colchicine is added. However, changes in lysosomal enzymatic activities remained biochemically undetectable. We conclude that: (a) the labile microtubular system does not seem indispensable for protein transport in the rough endoplasmic reticulum-Golgi area but may facilitate this step, perhaps by maintaining the spatial organization of this area; and (b) in the lacrimal gland, colchicine inhibits protein release not by acting on the steps of secretion following the secretory granule formation, but by acting chiefly on the steps preceding secretory granule formation, perhaps by making the secretory granules formed in its presence incapable of discharging their content.