A role for calmodulin in organelle membrane tubulation.

Membrane tubules of uniform diameter (60-80 nm) and variable lengths have been seen to extend from the main bodies of the Golgi complex, trans Golgi network (TGN), and endosomes. In the case of endosomes, these tubules appear to mediate membrane and receptor recycling events. Brefeldin A (BFA) is a potent drug that completely blocks coated vesicle formation from the Golgi complex and TGN, but at the same time causes the enhanced formation of membrane tubules from these same organelles. Recently, experiments have shown that calmodulin antagonists inhibit the transport of receptors out of endosomes, perhaps by inhibiting the formation of recycling tubules. Using the potent calmodulin-specific antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13), and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (C-1), we found that the recycling of transferrin from endosomes to the cell surface was significantly inhibited, resulting in the formation of enlarged endosomal vacuoles. In addition, these same calmodulin antagonists also potently inhibited the formation of BFA-stimulated membrane tubules from the Golgi complex, TGN, and endosomes. In the case of the Golgi complex, failure to form tubules resulted in the inhibition of BFA-stimulated retrograde transport to the endoplasmic reticulum. These results suggest that calmodulin is a general regulator of membrane tubulation and is capable of influencing the morphology of several organelles.     

The secretion inhibitor Exo2 perturbs trafficking of Shiga toxin between endosomes and the trans-Golgi network

The small-molecule inhibitor Exo2 {4-hydroxy-3-methoxy-(5,6,7,8-tetrahydrol[1]benzothieno[2,3-d]pyrimidin-4-yl)hydraz-one benzaldehyde} has been reported to disrupt the Golgi apparatus completely and to stimulate Golgi-ER (endoplasmic reticulum) fusion in mammalian cells, akin to the well-characterized fungal toxin BFA (brefeldin A). It has also been reported that Exo2 does not affect the integrity of the TGN (trans-Golgi network), or the direct retrograde trafficking of the glycolipid-binding cholera toxin from the TGN to the ER lumen. We have examined the effects of BFA and Exo2, and found that both compounds are indistinguishable in their inhibition of anterograde transport and that both reagents significantly disrupt the morphology of the TGN in HeLa and in BS-C-1 cells. However, Exo2, unlike BFA, does not induce tubulation and merging of the TGN and endosomal compartments. Furthermore, and in contrast with its effects on cholera toxin, Exo2 significantly perturbs the delivery of Shiga toxin to the ER. Together, these results suggest that the likely target(s) of Exo2 operate at the level of the TGN, the Golgi and a subset of early endosomes, and thus Exo2 provides a more selective tool than BFA for examining membrane trafficking in mammalian cells.     

Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin

We have studied the cytotoxicity of ricin in cells treated with brefeldin A (BFA), which dramatically disrupts the structure of the Golgi apparatus causing Golgi content and membrane to redistribute to the ER. BFA inhibits the cytotoxicity of ricin in Chinese hamster ovary, normal rat kidney, and Vero cells and abolishes the enhancement of ricin cytotoxicity by NH4Cl, nigericin, swainsonine, and tunicamycin or by a mutation in endosomal acidification. BFA protects cells from the cytotoxicities of modeccin and Pseudomonas toxin, but has no effect on the intoxication by diphtheria toxin. Pretreatment of BFA does not protect cells from ricin treatment in the absence of BFA. Our results suggest that ricin, modeccin, and Pseudomonas toxin share a common pathway of intracellular transport from endosomes to the Golgi region where they are released into the cytosol. In contrast, the lack of protection of Vero cells from diphtheria toxin by BFA indicates that diphtheria toxin is released from acidified endosomes without involving the Golgi region.     

Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes.

Brefeldin A (BFA) is a fungal metabolite that causes a redistribution of the stacked cisternae of the Golgi complex into the endoplasmic reticulum by inhibiting anterograde transport. We report that BFA also causes membrane tubules derived from the trans-Golgi network (TGN) to fuse with early endosomes. In the presence of BFA, a mannose-6-phosphate receptor (M6PR)-enriched tubular network rapidly forms from the TGN, not from the prelysosomal compartment, and can be labeled with endocytic tracers after only 5 min of uptake at either 20 degrees C or 37 degrees C, indicating that it is also functionally an early endosome. Formation of the TGN-early endosome network is microtubule dependent and may involve modification of membrane processes affected by microtubule-associated motor activity. Concomitant with the formation of the fused TGN-early endosome network, there is a greater than 5-fold increase in cell surface M6PRs. The data suggest that BFA has revealed a membrane transport cycle between the TGN and early endosomes, perhaps used for the secretion or delivery of molecules to the cell surface.     

The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A.

TGN38, a transmembrane glycoprotein predominantly localized to the trans-Golgi network, is utilized to study both the structure and function of the trans-Golgi network (TGN). The effects of brefeldin A (BFA) on the TGN were studied in comparison to its documented effects on the Golgi cisternae. During the first 30 min of BFA treatment, the TGN loses its cisternal structure and extends as tubules throughout the cytoplasm. By 60 min, it condenses into a stable structure surrounding the microtubule-organizing center. By electron microscopy, this structure appears as a population of large vesicles, and by immunolabeling, most of these vesicles contain TGN38. TGN38 cycles to the plasma membrane and back, which is shown by addition of TGN38 luminal domain antibodies directly to cell culture media. This results in rapid uptake of antibodies which label the TGN within 30 min, both in its native and BFA-induced conformation. A number of transmembrane proteins have been shown to take this cycling pathway, but TGN38 is unique in that it is the only one predominantly localized to the TGN. To investigate the cycling of TGN38, the endocytic pathway was labeled by internalization of Lucifer Yellow, and in the presence of BFA there was partial colocalization with TGN38. Further studies were carried out in which microtubules were depolymerized, resulting in dispersal of Golgi elements and inhibition of transport from endosomes to lysosomes. TGN38 cycling continues in the absence of microtubules. Taken together, these studies indicate that TGN38 returns from the plasma membrane via the endocytic pathway. We conclude that the TGN is structurally and functionally distinct from the Golgi cisternae, indicating that different molecules control membrane traffic from the Golgi cisternae and from the TGN.     

Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and endoplasmic reticulum (ER). To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes (EE), recycling endosomes, late endosomes and lysosomes. All cargos pass through EE, but may take different routes to the Golgi. Retromer-dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer-dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-mannose-6-phosphate receptor (CI-M6PR), which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CI-M6PR was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the EE, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer-dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB.     

BFA‐induced compartments from the Golgi apparatus and trans‐Golgi network/early endosome are distinct in plant cells

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5–10 μg ml^?1), BFA caused both the Golgi apparatus and trans‐Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY‐2 cells. Here we show that these BFA‐induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA‐induced Golgi‐ and TGN‐derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4‐64 co‐localized with the TGN‐derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose‐ and time‐dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4‐64 are mostly excluded from the SYP61‐positive BFA‐induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE‐derived BFA‐induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.     

Two lysosomal membrane proteins,LGP85 and LGP107, are delivered to late endosomes/lysosomes through different intracellular routes after exiting from the trans-Golgi network

Lysosomal membrane proteins are delivered from their synthesis site, the endoplasmic reticulum (ER) to late endosomes/lysosomes through the Golgi complex. It has been proposed that after leaving the Golgi they are transported either directly or indirectly (via the cell surface) to late endosomes/lysosomes. In the present study, we examined the transport routes taken by two structurally different lysosomal membrane proteins, LGP85 and LGP107, in rat 3Y1-B cells. Here we show that newly synthesized LGP85 and LGP107 are delivered to late endosomes/lysosomes via a direct route without passing through the cell surface. Interestingly, although LGP107 is delivered from the Golgi to early endosomes containing internalized horseradish peroxidase-conjugated transferrin (HRP-Tfn) en route to lysosomes, LGP85 does not pass through the HRP-Tfn-positive early endosomes. These results suggest, therefore, that LGP85 and LGP107 are sorted into distinct transport vesicles at the post-Golgi, presumably the trans-Golgi network (TGN), after which LGP85 is delivered directly to late endosomes/lysosomes, but significant fractions of LGP107 are targeted to early endosomes before transport to late endosomes/lysosomes. This study provides the first evidence that after exiting from the Golgi, LGP85 and LGP107 are targeted to late endosomes/lysosomes via a different pathway.     

In AtT20 and HeLa cells brefeldin A induces the fusion of tubular endosomes and changes their distribution and some of their endocytic properties

We have studied the effects of brefeldin A (BFA) on the tubular endosomes in AtT20 and HeLa cells (Tooze, J., and M. Hollinshead. 1991. J. Cell Biol. 115:635-653) by electron microscopy of cells labeled with three endocytic tracers, HRP, BSA-gold, and transferrin conjugated to HRP, and by immunofluorescence microscopy. For the latter we used antibodies specific for transferrin receptor, and, in the case of AtT20 cells, also antibodies specific for synaptophysin. In HeLa cells BFA at concentrations ranging from 1 micrograms to 10 micrograms/ml causes the dispersed patches of network of preexisting tubular early endosomes to be incorporated within 5 min into tubules approximately 50 nm in diameter but up to 40-50 microns long. These long, straight tubular endosomes are aligned along microtubules; they branch relatively infrequently to form an open network or reticulum extending from the cell periphery to the microtubule organizing center (MTOC). As the incubation with BFA is prolonged beyond 5 min, a steady state is reached in which many tubules are located in a dense network enclosing the centrioles, with branches extending in a more open network to the periphery. This effect of BFA, which is fully reversed within 15-30 min of washing out, is inhibited by pre-incubating the cells with sodium azide and 2-deoxy-D-glucose. In AtT20 cells BFA at 5 micrograms/ml or above causes the same sorts of changes, preexisting tubular endosomes are recruited into a more continuous endosomal network, and there is a massive accumulation of this network around the MTOC. Maintenance of the BFA-induced endosomal reticulum in both cell types is dependent upon the integrity of microtubules. In AtT20 cells BFA at 1 microgram/ml has no detectable effect on the early endosomal system but the Golgi stacks are converted to clusters of tubules and vesicles that remain in the region of the MTOC during prolonged incubations. Therefore, the Golgi apparatus in these cells is more sensitive to BFA than the early endosomes. The morphological evidence suggests that all the tubular early endosomes in BFA-treated HeLa and AtT20 cells are linked together in a single reticulum. Consistent with this, incubations as short as 1-3 min with 10 or 20 mg/ml HRP in the medium result in the entire endosomal reticulum in most of the BFA-treated cells being filled with HRP reaction product.(ABSTRACT TRUNCATED AT 400 WORDS)     

The morphology but not the function of endosomes and lysosomes is altered by brefeldin A

Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energy-dependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for > 12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.     

Targeting of membrane proteins to endosomes and lysosomes

The pathways involved in targeting membrane proteins to lysosomes are extraordinarily complex. Newly synthesized proteins in the ER are transported to the Golgi complex, and upon arrival at the trans Golgi network (TGN) are targeted either directly to endosomes, or first to the cell surface from where they can be rapidly internalized into the endocytic pathway for delivery to lysosomes. The routes to endosomes are specified by sorting motifs in the cytoplasmic tails of the proteins that are recognized at the TGN or plasma membrane. The molecular details of these processes are just emerging.     

Brefeldin A effects on the trans-Golgi network and Golgi bodies inBotryococcus braunii are not uniform during the cell cycle

Summary Using cryo-fixation and freeze-substitution electron microscopy, the effects of brefeldin A (BFA) on the structure of the trans-Golgi network (TGN), the endoplasmic reticulum (ER), and Golgi bodies in the unicellular green algaBotryococcus braunii were examined at various stages of the cell cycle. In the presence of BFA, all the TGNs of interphase and dividing cells aggregated to form a single tubular mass. In contrast, the TGNs decomposed just after cell division and disappeared during cell wall formation. Throughout the cell cycle, the TGN produced at least six kinds of vesicles, of which two were not formed in the presence of BFA: vesicles with a diameter of 200 nm and fibrillar substances, which formed in interphase cells; and vesicles with a diameter of 180–240 nm, which may participate in septum formation. In addition, the number of clathrin-coated vesicles attaching to the TGN decreased. In interphase cells, BFA induced the disassembly of Golgi bodies and an increase in the smooth-ER cisternae at the cis-side of Golgi bodies. This result may suggest the existence of retrograde transport from the Golgi bodies to the ER in the presence of BFA. These drastic structural changes in the Golgi bodies and the ER of interphase cells were not observed in BFA-treated dividing cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TGN trans-Golgi network     

Inhibition of membrane tubule formation and trafficking by isotetrandrine, an antagonist of G-protein-regulated phospholipase A2 enzymes

Previous studies have established a role for cytoplasmic phospholipase A(2) (PLA(2)) activity in tubule-mediated retrograde trafficking between the Golgi complex and the endoplasmic reticulum (ER). However, little else is known about how membrane tubule formation is regulated. This study demonstrates that isotetrandrine (ITD), a biscoclaurine alkaloid known to inhibit PLA(2) enzyme activation by heterotrimeric G-proteins, effectively prevented brefeldin A (BFA)-induced tubule formation from the Golgi complex and retrograde trafficking to the ER. In addition, ITD inhibited BFA-stimulated tubule formation from the trans-Golgi network and endosomes. ITD inhibition of the BFA response was potent (IC(50) approximately 10-20 microM) and rapid (complete inhibition with a 10-15-min preincubation). ITD also inhibited normal retrograde trafficking as revealed by the formation of nocodazole-induced Golgi mini-stacks at ER exit sites. Treatment of cells with ITD alone caused the normally interconnected Golgi ribbons to become fragmented and dilated, but cisternae were still stacked and located in a juxtanuclear position. These results suggest that a G-protein-binding PLA(2) enzyme plays a pivotal role in tubule mediated trafficking between the Golgi and the ER, the maintenance of the interconnected ribbons of Golgi stacks, and tubule formation from endosomes.     

Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38

Brefeldin A (BFA) has a dramatic effect on the morphology of the Golgi apparatus and induces a rapid redistribution of Golgi proteins into the ER (Lippincott-Schwartz, J., L. C. Yuan, J. S. Bonifacino, and R. D. Klausner. 1989. Cell. 56:801-813). To date, no evidence that BFA affects the morphology of the trans-Golgi network (TGN) has been presented. We describe the results of experiments, using a polyclonal antiserum to a TGN specific integral membrane protein (TGN38) (Luzio, J.P., B. Brake, G. Banting, K. E. Howell, P. Braghetta, and K. K. Stanley. 1990. Biochem. J. 270:97-102), which demonstrate that incubation of cells with BFA does induce morphological changes to the TGN. However, rather than redistributing to the ER, the majority of the TGN collapses around the microtubule organizing center (MTOC). The effect of BFA upon the TGN is (a) independent of protein synthesis, (b) fully reversible (c) microtubule dependent (as shown in nocodazole-treated cells), and (d) relies upon the hydrolysis of GTP (as shown by performing experiments in the presence of GTP gamma S). ATP depletion reduces the ability of BFA to induce a redistribution of Golgi proteins into the ER; however, it has no effect upon the BFA-induced relocalizations of the TGN. These data confirm that the TGN is an organelle which is independent of the Golgi, and suggest a dynamic interaction between the TGN and microtubules which is centered around the MTOC.     

Post-Golgi membrane traffic: brefeldin A inhibits export from distal Golgi compartments to the cell surface but not recycling

Recent studies using the fungal metabolite brefeldin A (BFA) have provided important insights into the dynamics and the organization of the ER/Golgi membrane system. Here we examined the effect of BFA on the functional integrity of the distal part of the secretory pathway, i.e., transport between trans-Golgi cisternae and the cell surface. To assay export via the constitutive pathway, we followed the movement of vesicular stomatitis virus (VSV) G glycoprotein that had been accumulated in the trans-Golgi network (TGN) by incubation of infected BHK-21 cells at 20 degrees C. Addition of BFA rapidly and reversibly inhibited cell surface transport of G protein. The block to secretion was not due to redistribution of externalized G protein to internal pools. It was also not due to collapse of TGN to the ER, since VSV G protein blocked in treated cells resided in compartments that were distinct from the ER/Golgi system. Similar effects were found with a bulk-flow marker: BFA blocked constitutive secretion of glycosaminoglycan chains that had been synthesized and sulfated in the trans-Golgi cisternae. To examine export via the regulated secretory pathway, we assayed secretion of [35S]SO4 labeled secretogranin II from PC12 cells, a marker that has been used to study secretory granule budding from the TGN (Tooze, S. A., U. Weiss, and W. B. Huttner. 1990. Nature [Lond.]. 347:207-208). BFA potently inhibited secretion of sulfated secretogranin II induced by K+ depolarization. Inhibition was at the level of granule formation, since BFA had no effect on regulated secretion from preformed granules. Taken together, the results suggest that BFA blocks export via both the constitutive and the regulated pathways. In contrast, endocytosis and recycling of VSV G protein were not blocked by BFA, consistent with previous studies that endocytosis is unaffected (Misumi, Y., Y. Misumi, K. Miki, A Takatsuki, G. Tamura, and Y. Ikehara. 1986. J. Biol. Chem. 261:11398-11403). These and earlier results suggest that the exo/endocytic pathway of mammalian cells consist of two similar but distinct endomembrane systems: an ER/Golgi system and a post-Golgi system. BFA prevents forward transport without affecting return traffic in both systems.     

Mepanipyrim,a novel inhibitor of pharmacologically induced Golgi dispersion

Mepanipyrim inhibited retrograde Golgi-to-ER trafficking induced by brefeldin A (BFA), nordihydroguaiaretic acid, clofibrate, and arachidonyltrifluoromethyl ketone in NRK and other types of cells, but did not inhibit anterograde trafficking of Golgi-resident proteins translocated to ER by BFA and newly synthesized VSV-G. However, mepanipyrim did not block the TGN38 dispersion induced by any of these compounds. Mepanipyrim acted on the Golgi, and swollen vesicular Golgi structures were formed and similar structures accumulated during rebuilding of the Golgi after BFA removal. These actions of mepanipyrim were readily reversed after its removal. Mepanipyrim did not stabilize microtubules, but prevented nocodazole-induced fragmentation and dispersion of the Golgi. These results suggest that the mepanipyrim-sensitive molecules participated in stabilizing the Golgi and its anchoring in the perinuclear region, and equally importantly, that the novel action of mepanipyrim may be used as a pharmacological tool for investigating membrane transport, Golgi membrane dynamics, and differentiation of the Golgi from TGN.     

Cycling of early Golgi proteins via the cell surface and endosomes upon lumenal pH disruption

The cis-Golgi protein GPP130 reversibly redistributes to endosomes upon pH disruption, but the identity of the endosomes and the involved cycling route are unknown. It is also unknown whether any other early Golgi proteins participate in this pathway. Here, we analyze GPP130 and the structurally related Golgi protein GP73. Unlike the TGN marker TGN38/46, GPP130 and GP73 colocalized in the early Golgi and redistributed to the ER after brefeldin A treatment. Nevertheless, after pH disruption by monensin, GPP130 and GP73 redistributed to endosomes containing redistributed TGN38/46, but not other endosomal markers. In common with TGN38/46, the redistribution involved transient appearance on the plasma membrane, and upon monensin washout, the proteins moved back to the Golgi along a microtubule- and PI3 kinase-independent route. Although GP73 did not associate with GPP130, its steady-state Golgi targeting was also mediated by a lumenal predicted coiled-coil stem domain. These findings indicate that at least two early Golgi proteins, each containing stem domain Golgi targeting determinants, cycle to the cell surface and back along the late endosome independent TGN38/46 pathway.     

An Exo2 derivative affects ER and Golgi morphology and vacuolar sorting in a tissue-specific manner in arabidopsis

We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the ADP ribosylation factor-GTP exchange factor (ARF-GEF) inhibitor brefeldin A (BFA), LG8 affects these post-Golgi events without causing the formation of BFA bodies. Up to concentrations of 50 μm, the effects of LG8 are reversible.     

Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes

BACKGROUND: In contrast to the intense attention devoted to research on intracellular sterol trafficking in animal cells, knowledge about sterol transport in plant cells remains limited, and virtually nothing is known about plant endocytic sterol trafficking. Similar to animals, biosynthetic sterol transport occurs from the endoplasmic reticulum (ER) via the Golgi apparatus to the plasma membrane. The vesicle trafficking inhibitor brefeldin A (BFA) has been suggested to disrupt biosynthetic sterol transport at the Golgi level. RESULTS: Here, we report on early endocytic sterol trafficking in Arabidopsis root epidermal cells by introducing filipin as a tool for fluorescent sterol detection. Sterols can be internalized from the plasma membrane and localize to endosomes positive for the early endosomal Rab5 GTPase homolog ARA6 fused to green fluorescent protein (GFP) (ARA6-GFP). Early endocytic sterol transport is actin dependent and highly BFA sensitive. BFA causes coaccumulation of sterols, endocytic markers like ARA6-GFP, and PIN2, a polarly localized presumptive auxin transport protein, in early endosome agglomerations that can be distinguished from ER and Golgi. Sterol accumulation in such aggregates is enhanced in actin2 mutants, and the actin-depolymerizing drug cytochalasin D inhibits sterol redistribution from endosome aggregations. CONCLUSIONS: Early endocytic sterol trafficking involves transport via ARA6-positive early endosomes that, in contrast to animal cells, is actin dependent. Our results reveal sterol-enriched early endosomes as targets for BFA interference in plants. Early endocytic sterol trafficking and recycling of polar PIN2 protein share a common pathway, suggesting a connection between plant endocytic sterol transport and polar sorting events.     

Tubulation of Golgi membranes in vivo and in vitro in the absence of brefeldin A

Recent in vivo studies with the fungal metabolite, brefeldin A (BFA), have shown that in the absence of vesicle formation, membranes of the Golgi complex and the trans-Golgi network (TGN) are nevertheless able to extend long tubules which fuse with selected target organelles. We report here that the ability to form tubules (> 7 microns long) could be reproduced in vitro by treatment of isolated, intact Golgi membranes with BFA under certain conditions. Surprisingly, an even more impressive degree of tubulation could be achieved by incubating Golgi stacks with an ATP-reduced cytosolic fraction, without any BFA at all. Similarly, tubulation of Golgi membranes in vivo occurred after treatment of cells with intermediate levels of NaN3 and 2-deoxyglucose. The formation of tubules in vitro, either by BFA treatment or low-ATP cytosol, correlated precisely with a loss of the vesicle-associated coat protein beta-COP from Golgi membranes. After removal of BFA or addition of ATP, membrane tubules served as substrates for the rebinding of beta-COP and for the formation of vesicles in vitro. These results provide support for the idea that a reciprocal relationship exists between tubulation and vesiculation (Klausner, R. D., J. G. Donaldson, and J. Lippincott-Schwartz. 1992. J. Cell Biol. 116:1071- 1080). Moreover, they show that tubulation is an inherent property of Golgi membranes, since it occurs without the aid of microtubules or BFA treatment. Finally the results indicate the presence of cytosolic factors, independent of vesicle-associated coat proteins, that mediate the budding/tubulation of Golgi membranes.