Effects of brefeldin A on the endocytic route. Redistribution of mannose 6-phosphate/insulin-like growth factor II receptors to the cell surface.

The effect of brefeldin A (BFA) on the trafficking of the mannose 6-phosphate/insulin-like growth factor II receptor within the endocytic route was analyzed. Treatment with BFA induced a redistribution of the receptor to the cell surface and increased both the binding and internalization of ligands 2-4-fold. The effect of BFA was dose- and time-dependent and reversible. Determinations of transport rates showed that BFA increases the internalization rate and the externalization rate of the receptor. This implies that the higher surface concentration is due to higher concentrations of receptor at the intracellular sites from where they recycle to the cell surface. The effect of BFA was additive to the redistribution induced by insulin-like growth factors I and II and was observed in all human and rodent cell lines analyzed. BFA increased also the cell surface expression of the Mr 46,000 mannose 6-phosphate receptor but not of the transferrin receptor. The results indicate that BFA interferes with the transport of mannose 6-phosphate receptors and affects the endocytosis of lysosomal enzymes by increasing the number of receptors available for recycling to the cell surface.     

Inhibition by brefeldin A of the specific B cell antigen presentation to MHC class II-restricted T cells

We have shown previously that specific Ag presentation is prevented by the inhibition of protein synthesis but nonspecific presentation is not. In the present paper, Ag presentation by Ag-specific B cells was examined for sensitivity to brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum. A20-HL B lymphoma expressing surface receptors specific for TNP was used as a B cell, and TNP-OVA was used as a specific Ag. The presence of BFA during pulsing of A20-HL cells with TNP-OVA inhibited the ability of the pulsed cells to stimulate 42-6A T cell clone, specific for OVA323-339 and Iad. The inhibition was not due to nonspecific toxicity of BFA, because the presence of BFA during pulsing of A20-HL cells with OVA323-339 did not affect their APC function. Ag binding to the receptor on A20-HL cells and internalization by the cells were observed in the presence of BFA. Thus, BFA might inhibit intracellular processing of specific Ag or intracellular complex formation of antigenic peptide from specific Ag with MHC class II molecules. Nonspecific Ag presentation by A20-HL cells, however, was resistant to BFA. A20-HL cells pulsed with OVA in the presence of BFA, even after fixation, could stimulate 42-6A cells to produce IL-2, although the IL-2 production was lower than that induced by A20-HL cells pulsed in the absence of BFA. These results suggest that the processing pathways for specific Ag and nonspecific Ag are different from each other, at least partly, in A20-HL cells.     

Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.     

Brefeldin a down-regulates the transferrin receptor in K562 cells

The fungal metabolite brefeldin A (BFA) induces profound alterations in the morphology of intracellular organelles. Although BFA promotes the formation of extensive tubular endosomal domains, our understanding of the effects of the antibiotic on vesicle traffic events associated with endocytosis is limited. Thus, alterations in the transferrin (Tf) receptor's endocytic/recycling pathway upon treatment of human erythroleukemia K562 cells with BFA were studied as a pharmacological response. Treatment of K562 cells with BFA caused a down-regulation in the number of cell surface Tf receptors. This effect is highly reminiscent of the well-known action of phorbol 12-myristate 13-acetate (PMA) on Tf receptor traffic in K562 cells. However, our results demonstrate that these two agents down-regulate the Tf receptor via different mechanisms. The effects of BFA and PMA were additive when K562 cells were incubated with both together. Using the In/Sur method, the endocytic rate constant for Tf internalization was determined and PMA was found to greatly enhance k_e, from 0.28 min^–1 to 0.43 min^–1, while BFA had little effect (K_e=0.20 min^–1). In contrast, BFA-treatment alters the exocytic rate constant for return of internalized receptors to the cell surface, with the largest effect exerted on a slow-release, monensin-sensitive, compartment. The sum of the endocytic and exocytic kinetic data support a model in which BFA and PMA down-regulate the Tf receptor in K562 cells by mechanistically distinct actions, with BFA targeting exocytic monensin-sensitive intracellular compartments and PMA acting to exert a profound influence on elements of receptor internalization.Abbreviations BFA brefeldin A - ARF ADP-ribosylation factor - HRP horseradish peroxidase - Tf transferrin - PMA phorbol 12-myristate 13-acetate - DMSO dimethyl sulfoxide - PBS phosphate-buffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin - FITC-Tf fluorescein isothiocyanate-labelled transferrin     

Cell-specific basolateral membrane sorting of the human liver Na(+)-dependent bile acid cotransporter

The human Na(+)-taurocholate cotransporting polypeptide (Ntcp) is located exclusively on the basolateral membrane of hepatocyte, but the mechanisms underlying its membrane sorting domain have not been fully elucidated. In the present study, a green fluorescent protein-fused human NTCP (NTCP-GFP) was constructed using the polymerase chain reaction and was stably transfected into Madin-Darby canine kidney (MDCK) and Caco-2 cells. Taurocholate uptake studies and confocal microscopy demonstrated that the polarity of basolateral surface expression of NTCP-GFP was maintained in MDCK cells but was lost in Caco-2 cells. Nocodazole (33 microM), an agent that causes microtubular depolymerization, partially disrupted the basolateral localization of NTCP-GFP by increasing apical surface expression to 33.5% compared with untreated cells (P < 0.05). Brefeldin A (BFA; 1-2 microM) disrupted the polarized basolateral localization of NTCP, but monensin (1.4 microM) had no affect on NTCP-GFP localization. In addition, low-temperature shift (20 degrees C) did not affect the polarized basolateral surface sorting of NTCP-GFP and repolarization of this protein after BFA interruption. In summary, these data suggest that the polarized basolateral localization of human NTCP is cell specific and is mediated by a novel sorting pathway that is BFA sensitive and monensin and low-temperature shift insensitive. The process may also involve microtubule motors.     

GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.     

Inhibition by brefeldin A of protein secretion from the apical cell surface of Madin-Darby canine kidney cells

The effect of brefeldin A (BFA) on total and polarized protein secretion was examined in MDCK cells. Increasing concentrations of BFA have increasingly inhibitory effects on total protein secretion. The total protein secretion was essentially unaffected by BFA at 0.5 microgram/ml. When the BFA concentration was increased to 10 and 30 micrograms/ml, the total protein secretion was reduced to about 70 and 25%, respectively, of the control level. Consistent with this effect on total protein secretion, the Golgi structure as revealed by C6-NBD-ceramide (a fluorescent ceramide analog) staining was essentially unaltered by 0.5 microgram/ml BFA, while 10 and 30 micrograms/ml BFA significantly dispersed the Golgi apparatus. When the polarity of protein secretion was examined, it was found that the ratio of proteins secreted from the apical to those from the basolateral surface was reduced from 1.5-2.0 to 0.4-0.7 by all three BFA concentrations. Furthermore, several proteins which are preferentially released from the apical surface were found to be released without apparent surface polarity, while several other proteins which were preferentially released from the basolateral surface were unaffected. This study suggests that BFA, at 0.5 microgram/ml, can selectively inhibit protein secretion from the apical surface without affecting total protein secretion. The inhibition of apical secretion results in enhanced protein secretion from the basolateral surface.     

Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites

The pathways of synaptic vesicle (SV) biogenesis and recycling are still poorly understood. We have studied the effects of Brefeldin A (BFA) on the distribution of several SV membrane proteins (synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rab3A) and on endosomal markers to investigate the relationship between SVs and the membranes with which they interact in cultured hippocampal neurons developing in isolation. In these neurons, SV proteins are detected as punctate immunoreactivity that is concentrated in axons but is also present in perikarya and dendrites. In the same neurons, the transferrin receptor, a well established marker of early endosomes, is selectively concentrated in perikarya and dendrites. In the perikaryal- dendritic region, BFA induced a dramatic tubulation of transferrin receptors as well as a cotubulation of the bulk of synaptophysin. Synaptotagmin, synaptobrevin, p29 and SV2 immunoreactivities retained a primarily punctate distribution. No tubulation of rab3A was observed. In axons, BFA did not produce any obvious alteration of the distribution of SV proteins, nor of peroxidase- or Lucifer yellow- labeled early endosomes. The selective effect of BFA on dendritic membranes suggests the existence of functional differences between the endocytic systems in dendrites and axons. Cotubulation of transferrin receptors and synaptophysin in the perikaryal-dendritic region is consistent with a functional interconnection between the traffic of SV proteins and early endosomes. The heterogeneous effects of BFA on SV proteins in this cell region indicates that SV proteins are differentially sorted upon exit from the TGN and are coassembled into SVs at the cell periphery.     

BIG1, a Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor,Is Required for GABA-Gated Cl– Influx Through Regulation of GABAA Receptor Trafficking

GABA_A receptors (GABA_ARs) mediate the majority of fast synaptic inhibition. Trafficking regulation and protein–protein interactions that maintain the appropriate number of GABA_ARs at the cell surface are considered to be important mechanisms for controlling the strength of synaptic inhibition. Here, we report that BIG1, a brefeldin A (BFA)-inhibited guanine nucleotide-exchange factor (GEF) which has a known role in vesicle trafficking, is a new binding partner of GABA_ARs. Treatment of neurons with BFA, an uncompetitive inhibitor of BIG1 GEF activity, or depletion of BIG1 by small RNA interference (siRNA) significantly decreased GABA_ARs at the neuronal surface and suppressed GABA-gated influx of chloride ions. Over-expression of HA-tagged BIG1-E793K, a dominant-negative mutant, also significantly decreased GABA_ARs at the neuronal surface, but had no effect on the total amount of GABA_ARs. Inhibition of GABA_AR endocytosis by muscimol increased both GABA_ARs and BIG1 at the neuronal surface in a time-dependent fashion, and this increase could be abolished by bicuculline. Finally, depletion of BIG1 by siRNA inhibited the muscimol-stimulated increase of GABA_ARs. Those data suggest an important function of BIG1 in trafficking of GABA_ARs to the cell surface through its GEF activity. Thus, we identify an important role of BIG1 in modulating GABA-gated Cl^? influx through the regulation of cell surface expression of GABA_ARs.     

布雷菲德菌素A调控IRE1-XBP1信号通路增强顺铂诱导GLC-82细胞凋亡的作用

长时间剧烈的内质网应激（endoplasmic reticulum stress,ERS）可启动ERS相关通路诱导细胞凋亡;而IREl-XBP1(肌醇需求蛋白1α-X盒结合蛋白1)为高度保守的ERS基本通路。它是最有前景的肿瘤治疗靶点之一。我们研究已证明,布雷菲德菌素A（brefeldin A,BFA）能增强顺铂（cis-dichlorodiamine platinum,CDDP）的肺癌细胞杀伤作用,但其分子机制尚不清楚。本研究证明了IRE1-XBP1通路在该协同效应中的作用。AnnexinV/PI双染结合流式细胞分析显示,与BFA或CDDP单独处理比较,BFA联合CDDP处理能增强人肺癌GLC-82细胞的凋亡。RT-qPCR和Western印迹揭示,与单独处理比较,BFA联合CDDP处理能增强GLC-82细胞的procaspase3转录本（mRNA）和蛋白质的表达,以及procaspase3的裂解激活,进一步证明了BFA联合CDDP处理可增强GLC-82细胞凋亡。最重要的是,RT-qPCR和Western印迹结果证明,与单独CDDP处理比较,BFA处理的GLC-82细胞中IRE1、XBP1水平明显上调（P < 0.05）,而BFA+CDDP处理的细胞中IRE1、XBP1水平进一步上调,超过BFA组（P < 0.01）。结果提示BFA和BFA+CDDP处理细胞可激活ERS的IRE1-XBP1通路。这些结果证明,BFA可通过激活ERS中的IRE1-XBP1通路增强顺铂诱导的肿瘤细胞凋亡。本研究结果为IREl-XBP1是颇具前景的肿瘤治疗靶点提供了新的证据。     

Induction of erythropoietin responsiveness in vitro by a distinct population of bone marrow cells.

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid 'burst-forming units' (BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.     

INDUCTION OF ERYTHROPOIETIN RESPONSIVENESS IN VITRO BY A DISTINCT POPULATION OF BONE MARROW CELLS

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid ‘burst-forming units’(BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.     

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.     

Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect

Whereas brefeldin A (BFA) protected a number of cell lines against the protein toxin ricin, two of the cell lines tested were not protected but rather sensitized to ricin by BFA. EM studies revealed that upon addition of BFA the Golgi stacks in cells which were protected against the toxin rapidly transformed into a characteristic tubulo-vesicular reticulum connected to the endoplasmic reticulum, and subcellular fractionation experiments showed that galactosyl transferase disappeared from the Golgi fractions where it was normally located. EM and subcellular fractionation also indicated that in contrast to the Golgi stacks, the trans-Golgi network (TGN) remained intact and that internalized ricin was still localized in the TGN both when BFA was added before and after the toxin. Thus, BFA does not prevent fusion of ricin-containing vesicles with the TGN, and unlike resident proteins in Golgi stacks, ricin is not transported back to ER upon treatment of cells with BFA. Two kidney epithelial cell lines, MDCK and PtK2, were not protected against ricin by BFA, and EM studies of MDCK cells revealed that BFA did not alter the morphology of the Golgi complex in these cells. Also, subcellular fractionation revealed that, in contrast to the other cell types tested, the localization of galactosyl transferase in the gradients was not affected by BFA treatment. The data show that there is a correlation between BFA-induced disassembly of the Golgi stacks and protection against ricin, and they demonstrate that the structural organization of the Golgi apparatus is affected by BFA to different extents in various cell lines.     

A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment.

Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers.     

Brefeldin-A enhancement of ricin A-chain immunotoxins and blockade of intact ricin, modeccin, and abrin

After binding, the protein toxins ricin, abrin, and modeccin are endocytosed and processed through the cell's vesicular system in a poorly understood fashion, prior to translocation to the cytosol. The role of the Golgi apparatus in toxin processing was studied using brefeldin-A (BFA), a fungal metabolite which blocks Golgi function. At concentrations that inhibit secretion of interleukin-2 (IL-2), BFA blocks ricin, modeccin, and abrin intoxication of a lymphocyte derived cell line (Jurkat). Paradoxically, BFA enhances the toxicity of two ricin A-chain immunotoxins targeted against distinct cell surface determinants. BFA concentrations which are optimal for immunotoxin enhancement are below those needed to affect ricin intoxication or IL-2 secretion. BFA blockade of ricin does not involve effects on ricin endocytosis, toxin translocation to the cytosol, or the enzymatic activity of toxin A-chain. In contrast, BFA has no effect on immunotoxin processing but does enhance the immunotoxin translocation step. It is concluded that: 1) intact Golgi function is required for holotoxin processing. 2) Intact Golgi function is not required for holotoxin translocation. 3) Golgi function is tightly linked to immunotoxin translocation. 4) BFA has effects on vesicular routing in addition to the block of Golgi function in secretion which has been reported.     

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.     

Biogenesis of insulin-responsive GLUT4 vesicles is independent of brefeldin A-sensitive trafficking

Insulin stimulates translocation of GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. As a significant amount of GLUT4 is localised to the TGN, independently of the biosynthetic pathway, one possibility is that trafficking via the TGN is important in either intracellular sequestration or insulin-dependent movement to the cell surface. In this study we have used immuno-electron microscopy to show that GLUT4 is localised to AP-1 vesicles in the TGN region in 3T3-L1 adipocytes. To dissect the role of this trafficking pathway we used brefeldin A (BFA) to disrupt AP-1 association with membranes. Despite a reorganisation of GLUT4 compartments following BFA treatment, the intracellular sequestration of GLUT4, and its insulin-dependent movement to the cell surface, was unaffected. BFA increased the half time of reversal of insulin-stimulated glucose transport from 17 to 30 min but did not prevent complete reversal. Furthermore, following reversal re-stimulation of glucose transport activity by insulin was not compromised. We conclude that under basal conditions GLUT4 cycles between the TGN and endosomes via the AP-1 pathway. However, neither this pathway, nor any other BFA-sensitive pathway, appears to play a major role in insulin-dependent recruitment of GLUT4 to the cell surface.     

Fission Yeast SCYL1/2 Homologue Ppk32: A Novel Regulator of TOR Signalling That Governs Survival during Brefeldin A Induced Stress to Protein Trafficking

Target of Rapamycin (TOR) signalling allows eukaryotic cells to adjust cell growth in response to changes in their nutritional and environmental context. The two distinct TOR complexes (TORC1/2) localise to the cell’s internal membrane compartments; the endoplasmic reticulum (ER), Golgi apparatus and lysosomes/vacuoles. Here, we show that Ppk32, a SCYL family pseudo-kinase, is a novel regulator of TOR signalling. The absence of ppk32 expression confers resistance to TOR inhibition. Ppk32 inhibition of TORC1 is critical for cell survival following Brefeldin A (BFA) induced stress. Treatment of wild type cells with either the TORC1 specific inhibitor rapamycin or the general TOR inhibitor Torin1 confirmed that a reduction in TORC1 activity promoted recovery from BFA induced stress. Phosphorylation of Ppk32 on two residues that are conserved within the SCYL pseudo-kinase family are required for this TOR inhibition. Phosphorylation on these sites controls Ppk32 protein levels and sensitivity to BFA. BFA induced ER stress does not account for the response to BFA that we report here, however BFA is also known to induce Golgi stress and impair traffic to lysosomes. In summary, Ppk32 reduce TOR signalling in response to BFA induced stress to support cell survival.