Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi network

A member of the A disintegrin and metalloproteinase domain with thrombospondin type-1 motifs (ADAMTS-4) protease family can efficiently cleave aggrecan at several sites detected in joints of osteoarthritic patients. Although recent studies have shown that removal of the prodomain of ADAMTS4 is critical for its ability to degrade aggrecan, the cellular mechanisms for its processing and trafficking remain unclear. In this study, by using both furin-specific inhibitor and RNA interference technique, we demonstrate that furin plays an important role in the intracellular removal of ADAMTS4 prodomain. Further, we demonstrate that proADAMTS4 can be processed by means of multiple furin recognition sites: (206)RPRR(209), (209)RAKR(212), or (211)KR(212). The processing of proADAMTS4 was completely blocked by brefeldin A treatment, suggesting that processing occurs in the trans-Golgi network. Indeed, ADAMTS4 is co-localized with furin in trans-Golgi network. Interestingly, the pro form of ADAMTS4, not its mature one, co-precipitates with furin, suggesting that furin physically interacts with the prodomain of ADAMTS-4. In addition, our evidence suggests that a furin-independent pathway may also contribute to the activation of ADAMTS4. These results indicate that the activation mechanism for ADAMTS4 can be targeted for therapeutical intervention against this enzyme.     

2A proteinase of human rhinovirus cleaves cytokeratin 8 in infected HeLa cells

Rhino- and enteroviruses encode two proteinases, 2A and 3C, which are responsible for the processing of the viral polyprotein and for cleavage of several cellular proteins. To identify further targets of the 2A proteinase of human rhinovirus serotype 2 (HRV2), an in vitro cleavage assay followed by two-dimensional electrophoresis was employed. Cytokeratin 8, a member of the intermediate filament group of proteins, was found to be proteolytically cleaved in vitro by the 2A proteinase of HRV2 and of coxsackievirus B4 and in vivo during HRV2 infection of HeLa cells. The cleavage results in removal of 14 amino acids from the N-terminal head domain of cytokeratin 8. However, other intermediate filament proteins (cytokeratins 7 and 18 and vimentin) were not cleaved in the course of the HRV2 infection. Compared with the processing of the eucaryotic translation initiation factors 4GI and 4GII, cleavage of cytokeratin 8 occurs late in the infection cycle at the time of the onset of the cytopathic effect.     

CLASPing microtubules at the trans-Golgi network

Microtubule (MT) arrays can be formed either from centrosomes or from noncentrosomal locations. In this issue of Developmental Cell, Efimov and colleagues report a role of CLASPs, the MT plus end-binding proteins, in MT formation from Golgi, implicating Golgi-originated MT arrays in efficient cell migration (Efimov et al., 2007).     

Enteroviral protease 2A directly cleaves dystrophin and is inhibited by a dystrophin-based substrate analogue

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy through unknown pathological mechanism(s). Dystrophin is a large extrasarcomeric cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy. In addition, we have recently shown that dystrophin is proteolytically cleaved by the Coxsackievirus protease 2A leading to functional impairment and morphological disruption. However, the mechanism of dystrophin cleavage and the exact cleavage site remained to be identified. Antibody epitope mapping of endogenous dystrophin indicated protease 2A-mediated cleavage at the site in the hinge 3 region predicted by a neural network algorithm (human, amino acid 2434; mouse, amino acid 2427). Using site-directed mutagenesis, peptide sequencing, and fluorescence resonance energy transfer assays with recombinant dystrophin, we demonstrate that this putative site in mouse and human dystrophin is a direct substrate for the Coxsackieviral protease 2A both in vitro and in vivo. The substrate analogue protease inhibitor z-LSTT-fmk was designed based on the dystrophin sequence that interacts with the protease 2A and was found to have an IC(50) of 550 nM in vitro. Dystrophin is the first cellular substrate of the enteroviral protease 2A that was identified using by a bioinformatic approach and for which the cleavage site was molecularly mapped within living cells.     

Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs

Here, we describe an in vitro assay that has permitted further characterization of a proteinase (called "ZP2-proteinase") that is released upon activation of ovulated mouse eggs and cleaves ZP2, one of three glycoproteins present in mouse zonae pellucidae. Results presented suggest that ZP2-proteinase readily diffuses through the zona pellucida within 5 min of activation of eggs by ionophore A23187 and carries out limited proteolysis of ZP2. Appearance of ZP2-proteinase is completely dependent upon activation of eggs, consistent with it being present in cortical granule exudate. The proteinase is insensitive to a wide variety of proteinase inhibitors, but is inhibited when either an anti-ZP2 monoclonal antibody or an Fab fragment of the antibody is bound to ZP2. Proteolysis occurs near the amino- or carboxy-terminus of ZP2, producing a 23,000 Mr glycopeptide(s) that remains attached to ZP2 by intramolecular disulfide bonds. HPLC fractionation of activated egg exudate suggests that ZP2-proteinase has an apparent Mr between 21,000 and 34,000. Proteolysis of ZP2 correlates with "hardening" of the zona pellucida following egg activation and, thus, may be responsible for one aspect of the zona reaction.     

Lipid-dependent protein sorting at the trans-Golgi network

In eukaryotic cells, the trans-Golgi network serves as a sorting station for post-Golgi traffic. In addition to coat- and adaptor-mediated mechanisms, studies in mammalian epithelial cells and yeast have provided evidence for lipid-dependent protein sorting as a major delivery mechanism for cargo sorting to the cell surface. The mechanism for lipid-mediated sorting is the generation of raft platforms of sphingolipids, sterols and specific sets of cargo proteins by phase segregation in the TGN. Here, we review the evidence for such lipid-raft-based sorting at the TGN, as well as their involvement in the formation of TGN-to-PM transport carriers. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Transition of galactosyltransferase 1 from trans-Golgi cisterna to the trans-Golgi network is signal mediated

The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various subcellular compartments or for secretion. Here we investigate beta1,4-galactosyltransferase 1 (galT) and alpha2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin-treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by colocalization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time-lapse videomicroscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from the cytoplasmic tail can be conferred to siaT, which leads to the rapid accumulation of the galT-siaT chimera in swollen vesicles upon monensin treatment. On the basis of these data, we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.     

Retrograde transport from endosomes to the trans-Golgi network

A subset of intracellular transmembrane proteins such as acid-hydrolase receptors, processing peptidases and SNAREs, as well as extracellular protein toxins such as Shiga toxin and ricin, undergoes 'retrograde' transport from endosomes to the trans-Golgi network. Here, we discuss recent studies that have begun to unravel the molecular machinery that is involved in this process. We also propose a central role for a 'tubular endosomal network' in sorting to recycling pathways that lead not only to the trans-Golgi network but also to different plasma-membrane domains and to specialized storage vesicles.     

AtVPS45 complex formation at the trans-Golgi network

The Sec1p family of proteins are thought to be involved in the regulation of vesicle fusion reactions through interaction with t-SNAREs (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) at the target membrane. AtVPS45 is a member of this family from Arabidopsis thaliana that we now demonstrate to be present on the trans-Golgi network (TGN), where it colocalizes with the vacuolar cargo receptor AtELP. Unlike yeast Vps45p, AtVPS45 does not interact with, or colocalize with, the prevacuolar t-SNARE AtPEP12. Instead, AtVPS45 interacts with two t-SNAREs, AtTLG2a and AtTLG2b, that show similarity to the yeast t-SNARE Tlg2p. AtTLG2a and -b each colocalize with AtVPS45 at the TGN; however, AtTLG2a is in a different region of the TGN than AtTLG2b by immunogold electron microscopy. Therefore, we propose that complexes containing AtVPS45 and either AtTLG2a or -b define functional subdomains of the TGN and may be required for different trafficking events. Among other Arabidopsis SNAREs, AtVPS45 antibodies preferentially coprecipitate AtVTI1b over the closely related isoform AtVTI1a, implying that AtVTI1a and AtVTI1b also have distinct functions within the cell. These data point to a functional complexity within the plant secretory pathway, where proteins encoded by gene families have specialized functions, rather than functional redundancy.     

Cargo trafficking between endosomes and the trans-Golgi network

The retrograde membrane transport pathways from endosomes to the trans-Golgi network (TGN) are now recognized as critical intracellular pathways to recycle and shuttle a selective subgroup of membrane proteins, including sorting receptors, membrane-bound enzymes, transporters, as well as providing an avenue for the intracellular transport of various bacterial toxins. Multiple pathways from endosomes to the TGN have now been defined which differ between the cargo transported and the machinery used. Here, we review advances in these pathways and the requirement for TGN organization, and also discuss the development of unbiased analytical approaches to quantitatively track cargo that use these endosome-to-TGN pathways.     

Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network

Clathrin-coated vesicles mediate endocytosis and transport between the trans-Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo and membranes. Two main types of clathrin adaptor act in TGN-endosome traffic: GGA proteins and the AP-1 complex. Here we characterize the relationship between GGA proteins, AP-1 and other TGN clathrin adaptors using live-cell and super-resolution microscopy in yeast. We present evidence that GGA proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PtdIns(4)P) levels at the TGN slow or uncouple AP-1 coat assembly from GGA coat assembly. Conversely, enhanced PtdIns(4)P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PtdIns(4)-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PtdIns(4)P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN.     

Phosphoinositides and membrane traffic at the trans-Golgi network

Cargo proteins moving along the secretory pathway are sorted at the TGN (trans-Golgi network) into distinct carriers for delivery to the plasma membrane or endosomes. Recent studies in yeast and mammals have shown that formation of these carriers is regulated by PtdIns(4)P. This phosphoinositide is abundant at the TGN and acts to recruit components required for carrier formation to the membrane. Other phosphoinositides are also present on the TGN, but the extent to which they regulate trafficking is less clear. Further characterization of phosphoinositide kinases and phosphatases together with identification of new TGN-associated phosphoinositide-binding proteins will reveal the extent to which different phosphoinositides regulate TGN trafficking, and help define the molecular mechanisms involved.     

Sorting within the regulated secretory pathway occurs in the trans-Golgi network

Bioactive peptides cleaved from the egg-laying hormone precursor in the bag cell neurons of Aplysia are sorted into distinct dense core vesicle classes (DCVs). Bag cell prohormone processing can be divided into two stages, an initial cleavage occurring in a late Golgi compartment, which is not blocked by monensin, and later cleavages that occur within DCVs and are blocked by monensin. Prohormone intermediates are sorted in the trans-Golgi network. The large soma-specific DCVs turn over, while the small DCVs are transported to processes for regulated release. Thus, protein trafficking differentially regulates the levels and localization of multiple biologically active peptides derived from a common prohormone.     

Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network

Autophagy is an intracellular bulk protein degradation system. Beclin is known to be involved in this process; however, its role is unclear. In this study, we showed that Beclin was co-immunoprecipitated with phosphatidylinositol (PtdIns) 3-kinase, which is also required for autophagy, suggesting that Beclin is a component of the PtdIns 3-kinase complex. Quantitative analyses using a cross-linker showed that all Beclin forms a complex with PtdIns 3-kinase, whereas ~50% of PtdIns 3-kinase remains free from Beclin. Indirect immunofluorescence microscopy demonstrated that the majority of Beclin and PtdIns 3-kinase localize to the trans-Golgi network (TGN). Some PtdIns 3-kinase is also distributed in the late endosome. These results suggest that Beclin and PtdIns 3-kinase control autophagy as a complex at the TGN.     

Bidirectional transport between the trans-Golgi network and the endosomal system

Abstract

The exchange of proteins and lipids between the trans-Golgi network (TGN) and the endosomal system requires multiple cellular machines, whose activities are coordinated in space and time to generate pleomorphic, tubulo-vesicular carriers that deliver their content to their target compartments. These machines and their associated protein networks are recruited and/or activated on specific membrane domains where they select proteins and lipids into carriers, contribute to deform/elongate and partition membrane domains using the mechanical forces generated by actin polymerization or movement along microtubules. The coordinated action of these protein networks contributes to regulate the dynamic state of multiple receptors recycling between the cell surface, endosomes and the TGN, to maintain cell homeostasis as exemplified by the biogenesis of lysosomes and related organelles, and to establish/maintain cell polarity. The dynamic assembly and disassembly of these protein networks mediating the exchange of membrane domains between the TGN and endosomes regulates cell-cell signalling and thus the development of multi-cellular organisms. Somatic mutations in single network components lead to changes in transport dynamics that may contribute to pathological modifications underlying several human diseases such as mental retardation.     

GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans -Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans -Golgi network.     

Syntaxin 6 functions in trans-Golgi network vesicle trafficking.

The specific transfer of vesicles between organelles is critical in generating and maintaining the organization of membrane compartments within cells. Syntaxin 6 is a recently discovered member of the syntaxin family, whose constituents are required components of several vesicle trafficking pathways. To better understand the function of syntaxin 6, we generated a panel of monoclonal antibodies that specifically recognize different epitopes of the protein. Immunoelectron microscopy shows syntaxin 6 primarily on the trans-Golgi network (TGN), where is partially colocalizes with the TGN adapter protein AP-1 on clathrin-coated membranes. Additional label is present on small vesicles in the vicinity of endosome-like structures. Immunoprecipitation of syntaxin 6 revealed that it is present in a complex or complexes with alpha-soluble NSF attachment protein, vesicle-associated membrane protein 2, or cellubrevin and a mammalian homologue of VPS45, which is a member of the sec1 family implicated in Golgi to prevacuolar compartment trafficking in yeast. We show that mammalian VPS45 is found in multiple tissues, is partially membrane associated, and is enriched in the Golgi region. Converging lines of evidence suggest that syntaxin 6 mediates a TGN trafficking event, perhaps targeting to endosomes in mammalian cells.     

Shrinkage and fragmentation of the trans-Golgi network in non-meristematic plant cells

Golgi products are exported from the trans-Golgi network (TGN) where they are sorted and packaged into secretory and clathrin-coated vesicles. We have examined TGN cisternae in Arabidopsis root columella cells and in maize basal endosperm transfer cells by electron microscopy/tomography. In these cell types, sizes of the TGN compartments decrease as they produce vesicles. After released from the Golgi, free TGN compartments continue to contract and they were seen to fragment into clusters of vesicles. The shrinkage of the plant TGN and its final disassembly suggest that the plant TGN is not a long-lasting organelle that is replenished regularly by membrane trafficking.Key words: trans-Golgi network, Golgi stack, root columella cell, basal endosperm transfer cell, secretory vesicle, clathrin-coated vesicle, electron tomographyThe TGN refers to a membranous compartment located on the trans-side of the Golgi stack, which sorts Golgi products according to their final destinations.¹ In plant cells, in which Golgi stacks are discrete and mobile, a trans-most Golgi cisterna transforms into a TGN cisterna and the TGN cisterna, later, peels away from the Golgi.² Once separated, movements of the Golgi and of the free TGN compartment are not coupled.^3,4Arabidopsis meristematic cells are small, averaging about 204 µm³ in volume.⁵ Golgi mobility is more restricted in small meristematic cells than in large vacuolated cells such as tobacco BY2 cells.^6,7 In these smaller cells, multiple TGN cisternae often remain associated with their original Golgi stacks, facilitating examination of the emergence of a TGN compartment and its subsequent maturation. We took advantage of the spatial proximity in Arabidopsis meristematic cells to delineate morphological features and protein localizations in the Golgi-associated (GA-) TGN and in free TGN.⁸ Our major findings include:(1) Transformation of a trans-Golgi cisterna into a GA-TGN cisterna involves the formation of secretory vesicle (SV) buds in the outer rim of the cisterna and a 30–35% reduction in cisternal membrane area.(2) RabA4b and phospatidylinositol-4-kinase β1 are associated with the GA-TGN and with the free TGN compartments, but are not associated with trans-Golgi cisternae.(3) Free TGN compartments fragment into SVs and clathrin-coated vesicles (CCVs) and into residual membrane pieces.In this addendum, electron microscopy/tomography analyses of the TGN in two non-meristematic cell types, namely Arabidopsis gravity-sensing root columella cells and maize basal endosperm transfer cells (BETCs), are reported. Formation and maturation of the TGN in these cell types agree with our findings from the meristematic TGN. Free TGN compartments are more abundant in these cell types than in the meristematic cells, facilitating examination of free TGN compartments. Withering and fragmentation of the free TGN compartments in these cell types suggest that the TGN is not a persistent organelle like the Golgi apparatus, which regularly revisits ER export sites to be sustained by the COPII vesicular transport system.     

Bidirectional transport between the trans-Golgi network and the endosomal system

The exchange of proteins and lipids between the trans-Golgi network (TGN) and the endosomal system requires multiple cellular machines, whose activities are coordinated in space and time to generate pleomorphic, tubulo-vesicular carriers that deliver their content to their target compartments. These machines and their associated protein networks are recruited and/or activated on specific membrane domains where they select proteins and lipids into carriers, contribute to deform/elongate and partition membrane domains using the mechanical forces generated by actin polymerization or movement along microtubules. The coordinated action of these protein networks contributes to regulate the dynamic state of multiple receptors recycling between the cell surface, endosomes and the TGN, to maintain cell homeostasis as exemplified by the biogenesis of lysosomes and related organelles, and to establish/maintain cell polarity. The dynamic assembly and disassembly of these protein networks mediating the exchange of membrane domains between the TGN and endosomes regulates cell-cell signalling and thus the development of multi-cellular organisms. Somatic mutations in single network components lead to changes in transport dynamics that may contribute to pathological modifications underlying several human diseases such as mental retardation.