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The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases., Graphical Abstract, Highlights • Trichomonas vaginalis phagolysosome consist of over 460 proteins. • Lysosomes are involved in secretion of virulence factors such as TvCP2. • N-glycosylation is required for lysosomal protein targeting. • T. vaginalis possesses homologs of mannose 6-phosphate receptor., In Brief Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in the parasitic protist Trichomonas vaginalis. We analyzed the phagolysosomal proteome that consists of over 460 proteins including important virulence factors. We demonstrated that glycosylation is involved in lysosomal protein targeting in T. vaginalis, which is unprecedented in parasitic protists. In addition to the classical secretory pathway, lysosomes are involved in unconventional protein secretion.

Choline phospholipids (PLs) such as phosphatidylcholine (PC) and 1-alkyl-2-acyl-sn-glycerophosphocholine are important components for cell membranes and also serve as a source of several lipid mediators. These lipids are biosynthesized in mammals in the final step of the CDP-choline pathway by the choline phosphotransferases choline phosphotransferase 1 (CPT1) and choline/ethanolamine phosphotransferase 1 (CEPT1). However, the contributions of these enzymes to the de novo biosynthesis of lipids remain unknown. Here, we established and characterized CPT1- and CEPT1-deficient human embryonic kidney 293 cells. Immunohistochemical analyses revealed that CPT1 localizes to the trans-Golgi network and CEPT1 to the endoplasmic reticulum. Enzyme assays and metabolic labeling with radiolabeled choline demonstrated that loss of CEPT1 dramatically decreases choline PL biosynthesis. Quantitative PCR and reintroduction of CPT1 and CEPT1 revealed that the specific activity of CEPT1 was much higher than that of CPT1. LC-MS/MS analysis of newly synthesized lipid molecular species from deuterium-labeled choline also showed that these enzymes have similar preference for the synthesis of PC molecular species, but that CPT1 had higher preference for 1-alkyl-2-acyl-sn-glycerophosphocholine with PUFA than did CEPT1. The endogenous level of PC was not reduced by the loss of these enzymes. However, several 1-alkyl-2-acyl-sn-glycerophosphocholine molecular species were reduced in CPT1-deficient cells and increased in CEPT1-deficient cells when cultured in 0.1% FBS medium. These results suggest that CEPT1 accounts for most choline PL biosynthesis activity, and that both enzymes are responsible for the production of different lipid molecular species in distinct organelles.

The Golgi apparatus (GA) is a cellular organelle that plays a critical role in the processing of proteins for secretion. Activation of G protein–coupled receptors at the plasma membrane (PM) induces the translocation of G protein βγ dimers to the GA. However, the functional significance of this translocation is largely unknown. Here, we study PM-GA translocation of all 12 Gγ subunits in response to chemokine receptor CXCR4 activation and demonstrate that Gγ9 is a unique Golgi-translocating Gγ subunit. CRISPR-Cas9–mediated knockout of Gγ9 abolishes activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), two members of the mitogen-activated protein kinase family, by CXCR4. We show that chemically induced recruitment to the GA of Gβγ dimers containing different Gγ subunits activates ERK1/2, whereas recruitment to the PM is ineffective. We also demonstrate that pharmacological inhibition of phosphoinositide 3-kinase γ (PI3Kγ) and depletion of its subunits p110γ and p101 abrogate ERK1/2 activation by CXCR4 and Gβγ recruitment to the GA. Knockout of either Gγ9 or PI3Kγ significantly suppresses prostate cancer PC3 cell migration, invasion, and metastasis. Collectively, our data demonstrate a novel function for Gβγ translocation to the GA, via activating PI3Kγ heterodimers p110γ-p101, to spatiotemporally regulate mitogen-activated protein kinase activation by G protein–coupled receptors and ultimately control tumor progression.

Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (β1) subunits, which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 μM, we detected BK activation in response to in vivo and in vitro CLR enrichment of myocytes. While a significant increase in myocyte CLR was achieved within 5 min of CLR in vitro loading, this brief CLR enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process. Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR enrichment. Moreover, CLR enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.

The Conserved Oligomeric Golgi (COG) complex, a multi-subunit vesicle tethering complex of the CATCHR (Complexes Associated with Tethering Containing Helical Rods) family, controls several aspects of cellular homeostasis by orchestrating retrograde vesicle traffic within the Golgi. The COG complex interacts with all key players regulating intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, and vesicular coats. In cells, COG deficiencies result in the accumulation of non-tethered COG-complex dependent (CCD) vesicles, dramatic morphological and functional abnormalities of the Golgi and endosomes, severe defects in N- and O- glycosylation, Golgi retrograde trafficking, sorting and protein secretion. In humans, COG mutations lead to severe multi-systemic diseases known as COG-Congenital Disorders of Glycosylation (COG-CDG). In this report, we review the current knowledge of the COG complex and analyze COG-related trafficking and glycosylation defects in COG-CDG patients., Graphical abstract Unlabelled Image, Highlights • Intracellular membrane trafficking and glycosylation. • COG complex structure and functions. • COG-CDGs: clinical presentation and molecular analysis.

In eukaryotic cells, protein sorting is a highly regulated mechanism important for many physiological events. After synthesis in the endoplasmic reticulum and trafficking to the Golgi apparatus, proteins sort to many different cellular destinations including the endolysosomal system and the extracellular space. Secreted proteins need to be delivered directly to the cell surface. Sorting of secreted proteins from the Golgi apparatus has been a topic of interest for over thirty years, yet there is still no clear understanding of the machinery that forms the post-Golgi carriers. Most evidence points to these post-Golgi carriers being tubular pleomorphic structures that bud from the trans-face of the Golgi. In this review, we present the background studies and highlight the key components of this pathway, we then discuss the machinery implicated in the formation of these carriers, their translocation across the cytosol, and their fusion at the plasma membrane.

Graphical abstract, ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules controlling major biological processes as diverse as DNA damage repair, transcriptional regulation, intracellular transport, immune and stress responses, cell survival and proliferation. Furthermore, enzymatic reactions of ADPr are central in the pathogenesis of many human diseases, including infectious conditions. By providing a review of ADPr signalling in bacterial systems, we highlight the relevance of this chemical modification in the pathogenesis of human diseases depending on host-pathogen interactions. The post-antibiotic era has raised the need to find alternative approaches to antibiotic administration, as major pathogens becoming resistant to antibiotics. An in-depth understanding of ADPr reactions provides the rationale for designing novel antimicrobial strategies for treatment of infectious diseases. In addition, the understanding of mechanisms of ADPr by bacterial virulence factors offers important hints to improve our knowledge on cellular processes regulated by eukaryotic homologous enzymes, which are often involved in the pathogenesis of human diseases.

Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K+ channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K+ channels, however, remain unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent nonglycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, nonglycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.

G protein–coupled receptors (GPCRs) are important modulators of synaptic functions. A fundamental but poorly addressed question in neurobiology is how targeted GPCR trafficking is achieved. Rab GTPases are the master regulators of vesicle-mediated membrane trafficking, but their functions in the synaptic presentation of newly synthesized GPCRs are virtually unknown. Here, we investigate the role of Rab43, via dominant-negative inhibition and CRISPR–Cas9–mediated KO, in the export trafficking of α2-adrenergic receptor (α2-AR) and muscarinic acetylcholine receptor (mAChR) in primary neurons and cells. We demonstrate that Rab43 differentially regulates the overall surface expression of endogenous α2-AR and mAChR, as well as their signaling, in primary neurons. In parallel, Rab43 exerts distinct effects on the dendritic and postsynaptic transport of specific α2B-AR and M3 mAChR subtypes. More interestingly, the selective actions of Rab43 toward α2B-AR and M3 mAChR are neuronal cell specific and dictated by direct interaction. These data reveal novel, neuron-specific functions for Rab43 in the dendritic and postsynaptic targeting and sorting of GPCRs and imply multiple forward delivery routes for different GPCRs in neurons. Overall, this study provides important insights into regulatory mechanisms of GPCR anterograde traffic to the functional destination in neurons.

GPR133 (ADGRD1), an adhesion G protein–coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF–CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM.

Background & Aims: Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve), which phosphorylates phosphatidylinositol-3-phosphate (PI3P). PI(3,5)P2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Methods: Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8), bile acids, and cigarette toxin was determined. Results: PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini. Conclusions: These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens, leading to the pathogenicity of acute pancreatitis. Keywords: Endosome, Pancreatitis, PIKfyve, Trypsin

Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)–Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell–matrix interface.

Connexins are membrane proteins that form GJ (gap junction) channels between adjacent cells. Cx43 (connexin 43), the most widely expressed member of the connexin family, has a rapid turnover rate, and its degradation involves both the lysosomal and ubiquitin–proteasome pathway. The goal of this work was to study the effects of geodiamolides, natural peptides from marine sponge that normally are involved with microfilament disruption, on connexin assembly or degradation in the plasma membrane. HTC (hepatocarcinoma cells) expressing Cx43–GFP (green fluorescent protein) were submitted to treatment with 200 nM geodiamolides A, B, H and I for 2 and 4 h. Microfilament distribution and the presence and size of GJ plaques were evaluated by laser scanning confocal microscopy. Among the four peptides tested, only Geo H (geodiamolide H) statistically enhanced the length of GJ plaques. Geodiamolide A also showed activity in the GJ plaque size; however, its effect was less pronounced. Treatment with Geo H could interfere with the delivery of connexins to the degradation structures, similar to proteasomal pathways, keeping the connexins assembled and accumulating GJ plaques. Further experiments, with the cells treated with Geo H, using the fungal antibiotic BFA (brefeldin A), were performed in order to uncouple events leading to GJ assembly from those related to GJ removal, since BFA is known to block protein trafficking within a fused ER (endoplasmic reticulum)/Golgi compartment. GJ plaques were drastically reduced after BFA/Geo H treatment, thus indicating that Geo H affects mainly the delivery pathway of Cx43 protein.

Insulin stimulates GLUT4 (glucose transporter 4) translocation in adipocytes and muscles. An emerging picture is that Rab10 could bridge the gap between the insulin signalling cascade and GLUT4 translocation in adipocytes. In the present study, two potential effectors of Rab10, GDI (guanine-nucleotide-dissociation inhibitor)-1 and GDI-2, are characterized in respect to their roles in insulin-stimulated GLUT4 translocation. It is shown that both GDI-1 and GDI-2 exhibit similar distribution to GLUT4 and Rab10 at the TGN (trans-Golgi network) and periphery structures. Meanwhile, GDI-1 clearly interacts with Rab10 with higher affinity, as shown by both immunoprecipitation and in vivo FRET (fluorescence resonance energy transfer). In addition, the participation of GDIs in GLUT4 translocation is illustrated when overexpression of either GDI inhibits insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Taken together, we propose that GDI-1 is preferentially involved in insulin-stimulated GLUT4 translocation through facilitating Rab10 recycling.

The hTRPC [human TRPC (canonical transient receptor potential)] family of non-selective cation channels is proposed to mediate calcium influx across the plasma membrane via PLC (phospholipase C)-coupled receptors. Heterologously expressed hTRPC3 and hTRPC7 have been localized at the cell surface; however, a large intracellular component has also been noted but not characterized. In the present study, we have investigated the intracellular pool in COS-7 cells and have shown co-localization with markers for both the TGN (trans-Golgi network) and the cis-Golgi cisternae by immunofluorescence microscopy. Addition of BFA (Brefeldin A) to cells expressing hTRPC3 or hTRPC7 resulted in the redistribution of the Golgi component to the endoplasmic reticulum, indicating that this pool is present in both the Golgi stack and the TGN. Expression of either TRPC3 or TRPC7, but not TRPC1 or the cell surface marker CD8, resulted in a 2–4-fold increase in secreted alkaline phosphatase in the extracellular medium. Based on these results, we propose that an additional function of these members of the hTRPC family may be to enhance secretion either by affecting transport through the Golgi stack or by increasing fusion at the plasma membrane.

Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI-anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis.

An understanding of the nature of immune protection and the role of immune effector products such as interferon-gamma (IFN-gamma) in the control of infectious disease is fundamental to the rational design of effective vaccines and immunotherapeutic reagents. Murine monoclonal and sheep polyclonal antibodies (mAbs and pAbs) to feline IFN-gamma (fIFN-gamma) were generated firstly to facilitate further research into the role of cellular immune responses in the control of feline infectious disease, and secondly to enable evaluation of the efficacy of novel immunotherapeutic approaches. A hybridoma clone, D9, secreting IgG1 antibodies was selected for expansion and the mAbs affinity purified in vitro. Polyclonal antibodies were raised in a sheep against recombinant fIFN-gamma and affinity purified. The sensitivity of the D9 mAb and the sheep anti-fIFN-gamma pAb was determined using an indirect fIFN-gamma enzyme-linked immunosorbent assay (ELISA) and immunoblots. These antibodies were assessed for their ability to detect the production of fIFN-gamma by specific feline T cell populations ex vivo following coculture with mitogen or feline leukaemia virus (FeLV) antigens for 4 h in the presence of the protein secretion inhibitor brefeldin A (BFA). Production of fIFN-gamma was evaluated using flow cytometry to simultaneously detect PE-labelled surface molecules and fluorescein isothiocyanate (FITC)-labelled intracellular fIFN-gamma. Using this approach, our initial studies revealed an upregulation in virus-specific fIFN-gamma-secreting CD4(+)T cells in the lymph nodes of FeLV latently infected cats.

Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure–activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants., In the present study a detailed characterization of a small molecule inhibitor of protein trafficking and gravitropic response is described. We also identified two Arabidopsis thaliana ecotypes that display resistance to this compound. The ecotypes and chemical provide useful tool to investigate protein trafficking.

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. The results of the present study implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.

Many G-protein-coupled receptors, including the alpha(1b)-adrenoceptor, form homo-dimers or oligomers. Mutation of hydrophobic residues in transmembrane domains I and IV alters the organization of the alpha(1b)-adrenoceptor oligomer, with transmembrane domain IV playing a critical role. These mutations also result in endoplasmic reticulum trapping of the receptor. Following stable expression of this alpha(1b)-adrenoceptor mutant, cell surface delivery, receptor function and structural organization were recovered by treatment with a range of alpha(1b)-adrenoceptor antagonists that acted at the level of the endoplasmic reticulum. This was accompanied by maturation of the mutant receptor to a terminally N-glycosylated form, and only this mature form was trafficked to the cell surface. Co-expression of the mutant receptor with an otherwise wild-type form of the alpha(1b)-adrenoceptor that is unable to bind ligands resulted in this wild-type variant also being retained in the endoplasmic reticulum. Ligand-induced cell surface delivery of the mutant alpha(1b)-adrenoceptor now allowed co-recovery to the plasma membrane of the ligand-binding-deficient mutant. These results demonstrate that interactions between alpha(1b)-adrenoceptor monomers occur at an early stage in protein synthesis, that ligands of the alpha(1b)-adrenoceptor can act as pharmacological chaperones to allow a structurally compromised form of the receptor to pass cellular quality control, that the mutated receptor is not inherently deficient in function and that an oligomeric assembly of ligand-binding-competent and -incompetent forms of the alpha(1b)-adrenoceptor can be trafficked to the cell surface by binding of a ligand to only one component of the receptor oligomer.

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.

Reduced pyridine nucleotide has been reported to enhance cell-free transfer of membrane material from a radiolabeled Golgi apparatus donor fraction from rat liver to an acceptor fraction consisting of inside-out plasma vesicles immobilized on nitrocellulose [(1992) Biochim. Biophys. Acta 1107, 131]. As part of a continuing effort to identify NADH-requiring enzymes in the Golgi apparatus which may be important to membrane trafficking, highly purified fractions of Golgi apparatus from rat liver were tested for their ability to oxidize NADH and the inhibition of the oxidation of NADH by brefeldin A. The isolated Golgi apparatus fractions were found to oxidize NADH with a specific activity comparable to that of the plasma membrane of rat liver. The activity was inhibited by brefeldin A and this inhibition was augmented by GDP. At near optimal concentrations of 7 microM brefeldin A and 1 microM GDP, the activity was > 90% inhibited. Brefeldin A inhibition of NADH oxidation by the Golgi apparatus was time-dependent and GDP appeared to accelerate the inhibition by brefeldin A.

Highlights • A. nidulans has a GBF/Gea and a BIG/Sec7 subfamily Golgi Arf1-GEFs, both essential. • The late Golgi Arf1-GEF mutant hypB5 conditionally blocks secretion. • Residue substitution in the early Golgi Arf1-GEF GeaA suppresses hypB5 and hypBΔ. • The mutation alters a GBF/Gea amino acid motif and shifts GeaA localization. • GeaA1 alone satisfies the eukaryotic requirement for two Golgi Arf1 GEFs., Golgi Arf1-guanine nucleotide exchange factors (GEFs) belong to two subfamilies: GBF/Gea and BIG/Sec7. Both are conserved across eukaryotes, but the physiological role of each is not well understood. Aspergillus nidulans has a single member of the early Golgi GBF/Gea-subfamily, geaA, and the late Golgi BIG/Sec7-subfamily, hypB. Both geaA and hypB are essential. hypB5 conditionally blocks secretion. We sought extragenic hypB5 suppressors and obtained geaA1. geaA1 results in Tyr1022Cys within a conserved GBF/Gea-specific S(Y/W/F)(L/I) motif in GeaA. This mutation alters GeaA localization. Remarkably, geaA1 suppresses hypBΔ, indicating that a single mutant Golgi Arf1-GEF suffices for growth.

TGN38 and TGN41 are isoforms of a monotopic integral membrane protein which recycles between the trans Golgi network (TGN) and the cell surface, but which, at steady state, is predominantly located in the TGN. Full-length and truncated versions of rat TGN38/41 have been expressed in monkey (COS) and human (Heb7a) cells under the control of the heavy metal inducible Metallothionein IIA promoter. This has allowed the regulated expression of TGN38/41 protein constructs to different levels in the transfected cells. These studies show that (i) controlled overexpression of TGN38/41 results in mislocalisation to parts of the endocytic pathway, (ii) a truncated version of TGN38/41, lacking the cytoplasmic domain, remains in the TGN, and (iii) there is a direct or indirect interaction between the cytoplasmic domain of TGN38/41 and γ-adaptin.