RNA interference-mediated silencing of the syntaxin 5 gene induces Golgi fragmentation but capable of transporting vesicles

It has been suggested that syntaxin 5 (Syx5) participates in vesicular transport. We examined the effects of Syx5 down-regulation on the morphology of the Golgi apparatus and the transport of vesicles in mammalian cells. Knockdown of the Syx5 gene resulted in Golgi fragmentation without changing the level of endoplasmic reticulum (ER)-resident proteins, other Golgi-SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors), and coatmer proteins. Strikingly, a major decrease in Syx5 expression barely affected the anterograde transport of vesicular stomatitis virus G (VSVG) protein to the plasma membrane. These results suggest that Syx5 is required for the maintenance of the Golgi structures but may not play a major role in the transport of vesicles carrying VSVG between the ER and the Golgi compartment.     

Maintenance of Golgi structure and function depends on the integrity of ER export.

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.     

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.     

Sec22 and Memb11 are v-SNAREs of the anterograde endoplasmic reticulum-Golgi pathway in tobacco leaf epidermal cells

Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.     

Role of Xklp3, a Subunit of the Xenopus Kinesin II Heterotrimeric Complex,in Membrane Transport between the Endoplasmic Reticulum and the Golgi Apparatus

The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.     

Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P- 450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.     

COG-7-deficient Human Fibroblasts Exhibit Altered Recycling of Golgi Proteins

Recently, we reported that two siblings presenting with the clinical syndrome congenital disorders of glycosylation (CDG) have mutations in the gene encoding Cog7p, a member of the conserved oligomeric Golgi (COG) complex. In this study, we analyzed the localization and trafficking of multiple Golgi proteins in patient fibroblasts under a variety of conditions. Although the immunofluorescent staining pattern of several Golgi proteins was indistinguishable from normal, the staining of endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC)-53 and the vesicular-soluble N-ethylmaleimide-sensitive factor attachment protein receptors GS15 and GS28 was abnormal, and the steady-state level of GS15 was greatly decreased. Retrograde transport of multiple Golgi proteins to the ER in patient fibroblasts via brefeldin A-induced tubules was significantly slower than occurs in normal fibroblasts, whereas anterograde protein trafficking was much less affected. After prolonged treatment with brefeldin A, several Golgi proteins were detected in clusters that colocalize with the microtubule-organizing center in patient cells. All of these abnormalities were normalized in COG7-corrected patient fibroblasts. These results serve to better define the role of the COG complex in facilitating protein trafficking between the Golgi and ER and provide a diagnostic framework for the identification of CDG defects involving trafficking proteins.     

Genes that allow yeast cells to grow in the absence of the HDEL receptor.

The ERD2 gene of Saccharomyces cerevisiae encodes the HDEL receptor that sorts ER proteins; it is essential for growth. In the absence of Erd2p the Golgi apparatus is both functionally and morphologically perturbed. Here we describe the isolation of four SED genes (suppressors of the erd2-deletion) which, when present in multiple copies, allow cells to grow in the absence of ERD2. The suppressed strains secrete the ER protein BiP and their internal membranes show a variety of morphological abnormalities. Sequence analysis indicates that all these SED genes encode membrane proteins: SED1 encodes a probable cell surface glycoprotein; SED2 is identical to SEC12, a gene required for the formation of ER-derived transport vesicles; SED4 encodes a protein whose cytoplasmic domain is 45% identical to that of Sec12p; SED3 is DPM1, the structural gene for dolichol-P-mannose synthase. We suggest that the absence of ERD2 causes an imbalance between membrane flow into and out of the Golgi apparatus, and that the SED gene products can compensate for this either by slowing transport from the ER or by stimulating vesicle budding from Golgi membranes.     

The Golgi apparatus and cell polarity: Roles of the cytoskeleton,the Golgi matrix,and Golgi membranes

Membrane trafficking plays a crucial role in cell polarity by directing lipids and proteins to specific subcellular locations in the cell and sustaining a polarized state. The Golgi apparatus, the master organizer of membrane trafficking, can be subdivided into three layers that play different mechanical roles: a cytoskeletal layer, the so-called Golgi matrix, and the Golgi membranes. First, the outer regions of the Golgi apparatus interact with cytoskeletal elements, mainly actin and microtubules, which shape, position, and orient the organelle. Closer to the Golgi membranes, a matrix of long coiled–coiled proteins not only selectively captures transport intermediates but also participates in signaling events during polarization of membrane trafficking. Finally, the Golgi membranes themselves serve as active signaling platforms during cell polarity events. We review here the recent findings that link the Golgi apparatus to cell polarity, focusing on the roles of the cytoskeleton, the Golgi matrix, and the Golgi membranes.     

Carbon tetrachloride suppresses ER-Golgi transport by inhibiting COPII vesicle formation on the ER membrane in the RLC-16 hepatocyte cell line

Carbon tetrachloride (CCl₄) causes hepatotoxicity in mammals, with its hepatocytic metabolism producing radicals that attack the intracellular membrane system and destabilize intracellular vesicle transport. Inhibition of intracellular transport causes lipid droplet retention and abnormal protein distribution. The intracellular transport of synthesized lipids and proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is performed by coat complex II (COPII) vesicle transport, but how CCl₄ inhibits COPII vesicle transport has not been elucidated. COPII vesicle formation on the ER membrane is initiated by the recruitment of Sar1 protein from the cytoplasm to the ER membrane, followed by that of the COPII coat constituent proteins (Sec23, Sec24, Sec13, and Sec31). In this study, we evaluated the effect of CCl₄ on COPII vesicle formation using the RLC-16 rat hepatocyte cell line. Our results showed that CCl₄ suppressed ER-Golgi transport in RLC-16 cells. Using a reconstituted system of rat liver tissue-derived cytoplasm and RLC-16 cell-derived ER membranes, CCl₄ treatment inhibited the recruitment of Sar1 and Sec13 from the cytosolic fraction to ER membranes. CCl₄-induced changes in the ER membrane accordingly inhibited the accumulation of COPII vesicle-coated constituent proteins on the ER membrane, as well as the formation of COPII vesicles, which suppressed lipid and protein transport between the ER and Golgi apparatus. Our data suggest that CCl₄ inhibits ER-Golgi intracellular transport by inhibiting COPII vesicle formation on the ER membrane in hepatocytes.     

Diacidic motifs influence the export of transmembrane proteins from the endoplasmic reticulum in plant cells

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins. We show that diacidic motifs perform a significant function in the export of transmembrane proteins to the Golgi apparatus, as mutations of these signals impede the efficient anterograde transport of multispanning, type II, and type I proteins. Furthermore, we demonstrate that diacidic motifs instigate the export of proteins that reside in the ER due to the lengths of their transmembrane domains. However, not all of the diacidic motifs in the cytosolic tails of the proteins studied were equally important in ER export. Transport of Golgi proteins was disrupted only by mutagenesis of specific diacidic signals, suggesting that the protein environment of these signals affects their function. Our findings indicate that diacidic ER export motifs are present and functional in plant membrane proteins and that they are dominant over transmembrane domain length in determining the export of proteins from the ER in plant cells.     

Both post-Golgi and intra-Golgi cycling affect the distribution of the Golgi phosphoprotein GPP130

Golgi phosphoprotein, GPP130, a cis Golgi protein, is representative of proteins cycling between the Golgi apparatus and endosomes in a pH-sensitive manner. The present qualitative data are insufficient to distinguish the relative contributions of Golgi and endosomal processes in regulating the cycling of such proteins. We have taken a quantitative approach to analyze GPP130 distribution in response to pH perturbation. We have used Shiga-like toxin B fragment, a protein that traffics from the cell surface and Golgi apparatus by the late endosomal bypass pathway, as a probe to highlight one aspect of GPP130 cycling and similarly the trafficking of tsO45-green fluorescent protein (GFP) between the Golgi apparatus and the plasma membrane to treat that aspect of GPP130 cycling in isolation. Overall, we conclude from quantitative analysis and simulations that treatment of HeLa cells with the pH perturbant, monensin, affects GPP130 cycling at several stages with effects on (i) intra-Golgi cycling, (ii) trans Golgi to endosome transport and (iii) endosome to Golgi transport. Our analysis indicates that the effect is greatest at the trans Golgi, the most acidic portion of the Golgi apparatus. In sum, multiple, regulated steps affect the trafficking of GPP130.     

Retargeting of viral glycoproteins into a non-exporting compartment during the myogenic differentiation of rat L6 cells

We have analysed protein trafficking during the differentiation of rat L6 myoblasts into myotubes. Different proteins were found to lose different amounts of their processing by the Golgi apparatus during the myogenic differentiation, indicating that they were transported to this organelle with differing efficiencies. In order to investigate the destination of the nonprocessed glycoproteins we analysed the behaviour of vesicular stomatitis virus (VSV) and Semliki Forest virus glycoproteins in the presence of Brefeldin A, which returns the enzymes of the Golgi apparatus to the ER. Such experiments indicated that during myogenesis a fraction of both glycoproteins was shunted into a compartment that did not participate recycling with the Golgi apparatus. Immunofluorescence studies with the mutant VSV tsO45 G protein suggested that this compartment was diffusively distributed. We investigated whether the cytoplasmic tail had a role in the myogenic transport modulation by analysing the behaviour of recombinant VSV G proteins. Exchanging the cytoplasmic tail or the tail plus the membrane anchor had no effect, suggesting that the luminal portion was responsible for the diverted transport. Taken together, the results suggest that during the myogenesis of L6 myoblasts, varying fractions of different viral glycoproteins were sorted from the ER into a specific compartment that did not recycle with the Golgi apparatus.     

The plant ER-Golgi interface: a highly structured and dynamic membrane complex

As compared with other eukaryotic cells, plants have developed an endoplasmic reticulum (ER)-Golgi interface with very specific structural characteristics. ER to Golgi and Golgi to ER transport appear not to be dependent on the cytoskeleton, and ER export sites have been found closely associated with Golgi bodies to constitute entire mobile units. However, the molecular machinery involved in membrane trafficking seems to be relatively conserved among eukaryotes. Therefore, a challenge for plant scientists is to determine how these molecular machineries work in a different structural and dynamic organization. This review will focus on some aspects of membrane dynamics that involve coat proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment receptor proteins), lipids, and lipid-interacting proteins.     

Rab6 interacts with the mint3 adaptor protein

The Rab6 GTPase regulates a retrograde transport route connecting endosomes and the endoplasmic reticulum (ER) via the Golgi apparatus. Recently it was shown that active (GTP-loaded) Rab6A regulates intracellular processing of the amyloid precursor protein (APP). To characterize the role of Rab6A in APP trafficking and to identify effector proteins of the active Rab6A protein, we screened a human placenta cDNA library using the yeast two-hybrid system. We isolated an interacting cDNA clone encoding part of the adaptor protein mint3. The interaction between Rab6A and mint3 is GTP-dependent and requires the complete phosphotyrosine-binding (PTB) domain of the mint protein, which also mediates the association with APP. By confocal microscopy we show that Rab6A, mint3 and APP co-localize at Golgi membranes in HeLa cells. Density gradient centrifugation of cytosolic extracts confirms a common distribution of these three proteins. Our data suggest that mint3 links Rab6A to APP traffic.     

Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) bind to a multi-SNAP receptor complex in Golgi membranes.

Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) are required for the binding of N-ethylmaleimide-sensitive fusion protein (NSF) to Golgi membranes and are, therefore, required for intra-Golgi transport. We report the existence of distinct alpha/beta-SNAP and gamma-SNAP-binding sites in Golgi membranes that appear to be part of the same receptor complex. Cross-linking studies with alpha-SNAP demonstrate that an integral membrane protein of between 30-40 kDa is the alpha-SNAP binding component of the multi-SNAP receptor complex. These data suggest that SNAPs function by independently binding to a multi-SNAP membrane-receptor complex, thereby activating them to serve as adaptors for the targeting of NSF.     

Retrograde transport of cholera toxin from the plasma membrane to the endoplasmic reticulum requires the trans-Golgi network but not the Golgi apparatus in Exo2-treated cells

Cholera toxin (CT) follows a glycolipid-dependent entry pathway from the plasma membrane through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER) where it is retro-translocated into the cytosol to induce toxicity. Whether access to the Golgi apparatus is necessary for transport to the ER is not known. Exo2 is a small chemical that rapidly blocks anterograde traffic from the ER to the Golgi and selectively disrupts the Golgi apparatus but not the TGN. Here we use Exo2 to determine the role of the Golgi apparatus in CT trafficking. We find that under the condition of complete Golgi ablation by Exo2, CT reaches the TGN and moves efficiently into the ER without loss in toxicity. We propose that even in the absence of Exo2 the glycolipid pathway that carries the toxin from plasma membrane into the ER bypasses the Golgi apparatus entirely.     

Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum

The tyrosine kinase Src is present on the Golgi membranes. Its role, however, in the overall function and organization of the Golgi apparatus is unclear. We have found that in a cell line called SYF, which lacks the three ubiquitous Src-like kinases (Src, Yes, and Fyn), the organization of the Golgi apparatus is perturbed. The Golgi apparatus is composed of collapsed stacks and bloated cisternae in these cells. Expression of an activated form of Src relocated the KDEL receptor (KDEL-R) from the Golgi apparatus to the endoplasmic reticulum. Other Golgi-specific marker proteins were not affected under these conditions. Because of the specific effect of Src on the location of KDEL-R, we tested whether protein transport between ER and the Golgi apparatus involves Src. Transport of Pseudomonas exotoxin, which is transported to the ER by binding to the KDEL-R is accelerated by inhibition or genetic ablation of Src. Protein transport from ER to the Golgi apparatus however, is unaffected by Src deletion or inhibition. We propose that Src has an appreciable role in the organization of the Golgi apparatus, which may be linked to its involvement in protein transport from the Golgi apparatus to the endoplasmic reticulum.     

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.     

A positive signal prevents secretory membrane cargo from recycling between the Golgi and the ER

The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward‐bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi‐to‐plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal‐deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tail‐anchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy‐ and Rab6‐dependent, and Rab6 inhibition accelerated signal‐deleted VSVG's transport to the cell surface. Our results extend the dynamic bi‐directional relationship between the Golgi and ER to include surface‐directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.