Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus

We have studied the role of a previously described tubulovesicular compartment near the cis-Golgi apparatus in endoplasmic reticulum (ER)-to-Golgi protein transport by light and immunoelectron microscopy in Vero cells. The compartment is defined by a 53-kDa transmembrane protein designated p53. When transport of the vesicular stomatitis virus strain ts045 G protein was arrested at 39.5 degrees C, the G protein accumulated in the ER but had access to the p53 compartment. At 15 degrees C, the G protein was exported from the ER into the p53 compartment which formed a compact structure composed of vesicular and tubular profiles in close proximity to the Golgi. Upon raising the temperature to 32 degrees C, the G protein migrated through the Golgi apparatus while the p53 compartment resumed its normal structure again. These results establish the p53 compartment as the 15 degrees C intermediate of the ER-to-Golgi protein transport pathway.     

Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.

Microtubule disruption has dramatic effects on the normal centrosomal localization of the Golgi complex, with Golgi elements remaining as competent functional units but undergoing a reversible "fragmentation" and dispersal throughout the cytoplasm. In this study we have analyzed this process using digital fluorescence image processing microscopy combined with biochemical and ultrastructural approaches. After microtubule depolymerization, Golgi membrane components were found to redistribute to a distinct number of peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed rapidly (within 15 minutes) to these sites after first moving through the ER. Prior to this redistribution, Golgi enzyme processing of proteins exported from the ER was inhibited and only returned to normal levels after Golgi enzymes redistributed to peripheral ER exit sites where Golgi stacks were regenerated. Experiments examining the effects of microtubule disruption on the membrane pathways connecting the ER and Golgi suggested their potential role in the dispersal process. Whereas clustering of peripheral pre-Golgi elements into the centrosomal region failed to occur after microtubule disruption, Golgi-to-ER membrane recycling was only slightly inhibited. Moreover, conditions that impeded Golgi-to-ER recycling completely blocked Golgi fragmentation. Based on these findings we propose that a slow but constitutive flux of Golgi resident proteins through the same ER/Golgi cycling pathways as ERGIC-53 underlies Golgi Dispersal upon microtubule depolymerization. Both ERGIC-53 and Golgi proteins would accumulate at peripheral ER exit sites due to failure of membranes at these sites to cluster into the centrosomal region. Regeneration of Golgi stacks at these peripheral sites would re-establish secretory flow from the ER into the Golgi complex and result in Golgi dispersal.     

Cell fractionation analysis of human CD8 glycoprotein transport between endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells

We have set up an analytical cell fractionation procedure to dissect, by a non-morphological method, the anterograde transport of proteins from endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells. Using this procedure after pulse-chase labelling of cells expressing human CD8 glycoprotein, we obtained results that: (1) support the view that the intermediate compartment is a distinct station in the export from the endoplasmic reticulum to the Golgi complex; and (2) strongly suggests that the O -glycosylation process starts after the intermediate compartment, presumably in the cis -Golgi complex.     

Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond

Significant advances have been made in recent years that have increased our understanding of the trafficking to and from membranes that are functionally linked to the Golgi apparatus in plants. New routes from the Golgi to organelles outside the secretory pathway are now being identified, revealing the importance of the Golgi apparatus as a major sorting station in the plant cell. This review discusses our current perception of Golgi structure and organization as well as the molecular mechanisms that direct traffic in and out of the Golgi.     

KDEL and KKXX retrieval signals appended to the same reporter protein determine different trafficking between endoplasmic reticulum,intermediate compartment,and Golgi complex

Many endoplasmic reticulum (ER) proteins maintain their residence by dynamic retrieval from downstream compartments of the secretory pathway. In previous work we compared the retrieval process mediated by the two signals, KKMP and KDEL, by appending them to the same neutral reporter protein, CD8, and found that the two signals determine a different steady-state localization of the reporter. CD8-K (the KDEL-bearing form) was restricted mainly to the ER, whereas CD8-E19 (the KKMP-bearing form) was distributed also to the intermediate compartment and Golgi complex. To investigate whether this different steady-state distribution reflects a difference in exit rates from the ER and/or in retrieval, we have now followed the first steps of export of the two constructs from the ER and their trafficking between ER and Golgi complex. Contrary to expectation, we find that CD8-K is efficiently recruited into transport vesicles, whereas CD8-E19 is not. Thus, the more restricted ER localization of CD8-K must be explained by a more efficient retrieval to the ER. Moreover, because most of ER resident CD8-K is not O-glycosylated but almost all CD8-E19 is, the results suggest that CD8-K is retrieved from the intermediate compartment, before reaching the Golgi, where O-glycosylation begins. These results illustrate how different retrieval signals determine different trafficking patterns and pose novel questions on the underlying molecular mechanisms.     

ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi

The temperature and ATP dependence of transport of the vesicular stomatitis virus strain ts045 G protein from the endoplasmic reticulum (ER) to an early Golgi compartment containing mannosidase I was studied in the mutant Chinese hamster ovary cell clone 15B. Appearance of G protein containing the Man5GlcNAc2 oligosaccharide species occurred after a shift to the permissive temperature with a lag period of 5 min and without detectable formation of the intermediate Man7GlcNAc2 and Man6GlcNAc2 species. Two biochemically distinct transport steps were detected during transport from the ER to the Golgi. An initial step is temperature sensitive, thermoreversible, and requires a high threshold of cellular ATP for maximal rate of transport (80% of the normal cellular ATP pool). Export from the ER is inhibited at 65% of the normal cellular ATP pool. Prolonged incubation at reduced levels of cellular ATP or at the restrictive temperature resulted in the accumulation of G protein in either the Man8GlcNAc2 species or the Man7GlcNAc2 and Man6GlcNAc2 species, respectively. Reversal of the temperature-sensitive block is ATP coupled. A second step is insensitive to incubation at the restrictive temperature and proceeds efficiently when the cellular ATP pool is reduced to 20% of the control. G protein accumulates at this intermediate step during prolonged incubation at 15 degrees C. The data suggest a functional division of processes required for transport of protein between the ER and Golgi compartments. The two steps may reflect the export (budding) and delivery (fusion) of proteins through vesicular trafficking between the ER and Golgi.     

Bidirectional membrane traffic between the endoplasmic reticulum and Golgi apparatus

Membrane traffic between the endoplasmic reticulum and Golgi apparatus is a highly regulated process that uses distinct anterograde and retrograde pathways. These pathways link two organelles that together function as a dynamic membrane system specialized for the biosynthesis and sorting of membrane to be used throughout the cell. The nature and underlying biochemical control of membrane transport along these pathways is thought to be tied to a common regulatory system involving assembly and disassembly of cytosolic proteins on membranes.     

Immunocytochemical analysis of Uukuniemi virus budding compartments: role of the intermediate compartment and the Golgi stack in virus maturation.

Previous studies have suggested that Uukuniemi virus, a bunyavirus, matures at the membranes of the Golgi complex. In this study we have employed immunocytochemical techniques to analyze in detail the budding compartment(s) of the virus. Electron microscopy of infected BHK-21 cells showed that virus particles are found in the cisternae throughout the Golgi stack. Within the cisternae, the virus particles were located preferentially in the dilated rims. This would suggest that virus budding may begin at or before the cis Golgi membranes. The virus budding compartment was studied further by immunoelectron microscopy with a pre-Golgi intermediate compartment marker, p58, and a Golgi stack marker protein, mannosidase II (ManII). Virus particles and budding virus were detected in ManII-positive Golgi stack membranes and, interestingly, in both juxtanuclear and peripheral p58-positive elements of the intermediate compartment. In cells incubated at 15 degrees C the nucleocapsid and virus envelope proteins were seen to accumulate in the intermediate compartment. Immunoelectron microscopy demonstrated that at 15 degrees C the nucleocapsid is associated with membranes that show a characteristic distribution and tubulo-vesicular morphology of the pre-Golgi intermediate compartment. These membranes contained virus particles in the lumen. The results indicate that the first site of formation of Uukuniemi virus particles is the pre-Golgi intermediate compartment and that virus budding continues in the Golgi stack. The results raise questions about the intracellular transport pathway of the virus particles, which are 100 to 120 nm in diameter and are therefore too large to be transported in the 60-nm-diameter vesicles postulated to function in the intra-Golgi transport. The distribution of the virus in the Golgi stack may imply that the cisternae themselves have a role in the vectorial transport of virus particles.     

Evidence against an essential role of COPII-mediated cargo transport to the endoplasmic reticulum-Golgi intermediate compartment in the formation of the primary membrane of vaccinia virus

Vaccinia virus assembles two distinct lipoprotein membranes. The primary membrane contains nonglycosylated proteins, appears as crescents in the cytoplasm, and delimits immature and mature intracellular virions. The secondary or wrapping membrane contains glycoproteins, is derived from virus-modified trans-Golgi or endosomal cisternae, forms a loose coat around some intracellular mature virions, and becomes the envelope of extracellular virions. Although the mode of formation of the wrapping membrane is partially understood, we know less about the primary membrane. Recent reports posit that the primary membrane originates from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). According to this model, viral primary membrane proteins are cotranslationally inserted into the ER and accumulate in the ERGIC. To test the ERGIC model, we employed Sar1(H79G), a dominant negative form of the Sar1 protein, which is an essential component of coatomer protein II (COPII)-mediated cargo transport from the ER to the ERGIC and other post-ER compartments. Overexpression of Sar1(H79G) by transfection or by a novel recombinant vaccinia virus with an inducible Sar1(H79G) gene resulted in retention of ERGIC 53 in the ER but did not interfere with localization of viral primary membrane proteins in factory regions or with formation of viral crescent membranes and infectious intracellular mature virions. Wrapping of intracellular mature virions and formation of extracellular virions did not occur, however, because some proteins that are essential for the secondary membrane were retained in the ER as a consequence of Sar1(H79G) overexpression. Our data argue against an essential role of COPII-mediated cargo transport and the ERGIC in the formation of the viral primary membrane. Instead, viral membranes may be derived directly from the ER or by a novel mechanism.     

Recycling of proteins between the endoplasmic reticulum and Golgi complex.

Several lines of investigation have shown that protein transport from the endoplasmic reticulum to the Golgi is more complex than previously imagined. Dynamic sorting of both membrane and soluble proteins is now believed to occur on the cis side of the Golgi apparatus with some proteins returning to the endoplasmic reticulum while others travel onwards.     

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.     

Inheritance of the endoplasmic reticulum and Golgi apparatus

Yeast and mammalian cells use a variety of different mechanisms to ensure that the endoplasmic reticulum and Golgi apparatus are inherited by both daughter cells on cell division. In yeast, endoplasmic reticulum inheritance involves both active microtubule and passive actin-based mechanisms, while the Golgi is transported into the forming daughter cell by an active actin-based mechanism. Animal cells actively partition the endoplasmic reticulum and Golgi apparatus, but association with the mitotic spindle-rather than the actin cytoskeleton-appears to be the mechanism     

Crossing the divide--transport between the endoplasmic reticulum and Golgi apparatus in plants

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.     

Impact on the endoplasmic reticulum and Golgi apparatus of turnip mosaic virus infection

The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K(2). TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K(2) fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K(2) vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K(2) vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.     

Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex

The transfer of newly synthesized membrane proteins moving from the rough endoplasmic reticulum (RER) to the Golgi complex has been studied by electron microscopy in HEp-2 cells transfected with cDNAs for chimeric proteins. These proteins consist of a reporter enzyme, horseradish peroxidase (HRP), anchored to the transmembrane domains of two integral membrane proteins, the transferrin receptor and sialyl- transferase. The chimeras are distributed throughout the nuclear envelope, RER, vesicular tubular clusters (VTCs) and a network of tubules in the cis-Golgi area. At 20 degrees C tubules containing chimera connect the RER to the VTCs and to the cis-Golgi network. On transfer to 37 degrees C in the presence of dithiothreitol (DTT), the chimeras are seen to move from the RER and through the Golgi stack. With this temperature shift the direct connections with the RER are lost and free vesicles form; some of these vesicles contain HRP reaction product which is much more concentrated than in the adjacent RER while others lack reaction product entirely. In cells expressing SSHRPKDEL, DAB reaction product remains distributed throughout the RER, the VTCs, and the cis-Golgi network for prolonged periods in the presence of DTT and almost all of the vesicles which form at 37 degrees C are DAB-positive. Together these observations demonstrate that all three chimeras are transported from the RER to the cis-Golgi in free, 40-60-nm vesicles at 37 degrees C. They also suggest that the retrograde traffic which carries SSHRPKDEL back to the RER is probably mediated by vesicles with a similar morphology but which, in cells expressing membrane-anchored chimeras, lack detectable reaction product.     

Mutations blocking the transport of the influenza virus hemagglutinin between the rough endoplasmic reticulum and the Golgi apparatus.

Mutants ts1 and ts227 of fowl plague virus have a temperature-sensitive defect in the transport of the hemagglutinin from the rough endoplasmic reticulum to the Golgi apparatus. The primary structure of the hemagglutinin of the mutants and of a number of revertants derived from them has been analysed by nucleotide sequencing. The transport block of the hemagglutinin of ts227 can be attributed to a single amino acid exchange. It involves the replacement of aspartic acid at position 457 by asparagine thereby introducing a new glycosylation site which appears to be located in a cryptic position in the lower part of the hemagglutinin stalk. Attachment of carbohydrate to this site is temperature-dependent. At permissive temperature only a small fraction of the monomers (approximately 30%) is glycosylated in this position, whereas at nonpermissive temperature this is the case with all subunits. The data suggest that under the latter conditions the new oligosaccharide interferes by steric hindrance with the trimerization of the hemagglutinin. The hemagglutinin of ts1 has an essential amino acid exchange at position 275 where serine is replaced by glycine. This substitution may increase the flexibility of the molecule in the hinge region between the globular domain and the stalk. The exchange of a conserved glutamic acid residue at position 398 that is involved in the interaction between different monomers contributes also to the structural instability of the ts1 hemagglutinin. These observations support the notion that the transport of the hemagglutinin from the rough endoplasmic reticulum to the Golgi apparatus depends on trimer assembly.     

Implication of ZW10 in membrane trafficking between the endoplasmic reticulum and Golgi

ZW10, a dynamitin-interacting protein associated with kinetochores, is known to participate directly in turning off of the spindle checkpoint. In the present study, we show that ZW10 is located in the endoplasmic reticulum as well as in the cytosol during interphase, and forms a subcomplex with RINT-1 (Rad50-interacting protein) and p31 in a large complex comprising syntaxin 18, an endoplasmic reticulum-localized t-SNARE implicated in membrane trafficking. Like conventional syntaxin-binding proteins, ZW10, RINT-1 and p31 dissociated from syntaxin 18 upon Mg(2+)-ATP treatment in the presence of NSF and alpha-SNAP, whereas the subcomplex was not disassembled. Overexpression, microinjection and knockdown experiments revealed that ZW10 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. The present results disclose an unexpected role for a spindle checkpoint protein, ZW10, during interphase.     

Ras signalling on the endoplasmic reticulum and the Golgi

Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.     

Retinoid modulation of cell-free membrane transfer between endoplasmic reticulum and Golgi apparatus

Cell-free transfer of radiolabeled membrane proteins from part-rough, part-smooth transitional elements of the endoplasmic reticulum to Golgi apparatus immobilized to nitrocellulose in the presence of nucleoside triphosphate, an ATP-regenerating system and a cytosol fraction was promoted by retinol. At an optimum concentration of 1 microgram/ml, the rate and amount of transfer was approximately doubled over 1 to 2 h of incubation in the cell-free system. The transition vesicles induced to form in the cell-free system were concentrated by preparative free-flow electrophoresis in order to study separately the steps of vesicle formation from transitional endoplasmic reticulum and the steps of vesicle fusion with Golgi apparatus. The retinol effect was on vesicle formation as evidenced by an approx. 2-fold increase in transition vesicle numbers, as determined by electron microscope morphometry, and amount from protein determinations on the isolated fractions enriched in transition vesicles. The retinol response in the complete transfer could be eliminated by addition of concentrated cytosol, including cytosol depleted of retinol. An interaction of retinol with some component of the vesicle formation process, possibly involving guanine nucleotides, is indicated.