Direct and GTP-dependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature secretory granules

ADP-ribosylation factor 1 (ARF1) mediates clathrin coat formation on PC12 immature secretory granules (ISGs). We have used two approaches to investigate whether ARF1 interacts directly with the clathrin adaptor protein, AP-1. Using an in vitro recruitment assay and co-immunoprecipitation, we could isolate an AP-1.ARF1 complex. Then we used a site-directed photocross-linking approach to determine the components that act downstream of ARF1 in clathrin coat formation on ISGs. Myristoylated ARF1, with a photolabile phenylalanine analogue incorporated into its putative effector domain (switch 1), showed a specific, GTP-dependent interaction with both the gamma- and beta-adaptin subunits of AP-1 on ISGs. These experiments provide evidence for a direct interaction of ARF1 with AP-1. On mature secretory granules myristoylated ARF1 does not bind, and hence clathrin coat formation cannot be initiated, supporting the hypothesis that molecules involved in coat recruitment are removed during ISG maturation.     

Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation.

The composition of secretory granules in neuroendocrine and endocrine cells is determined by two sorting events; the first in the trans-Golgi complex (TGN), the second in the immature secretory granule (ISG). Sorting from the ISG, which may be mediated by the AP-1 type adaptor complex and clathrin-coated vesicles, occurs during ISG maturation. Here we show that furin, a ubiquitously expressed, TGN/endosomal membrane endoprotease, is present in the regulated pathway of neuroendocrine cells where it is found in ISGs. By contrast, TGN38, a membrane protein that is also routed through the TGN/endosomal system does not enter ISGs. Furin, however, is excluded from mature secretory granules, suggesting that the endoprotease is retrieved from the clathrin-coated ISGs. Consistent with this, we show that the furin cytoplasmic domain interacts with AP-1, a component of the TGN/ISG-localized clathrin sorting machinery. Interaction between AP-1 and furin is dependent on phosphorylation of the enzyme's cytoplasmic domain by casein kinase II. Finally, in support of a requirement for the phosphorylation-dependent association of furin with AP-1, expression of furin mutants that mimic either the phosphorylated or unphosphorylated forms of the endoprotease in AtT-20 cells demonstrates that the integrity of the CKII sites is necessary for removal of furin from the regulated pathway.     

Homotypic Fusion of Immature Secretory Granules During Maturation Requires Syntaxin 6

Homotypic fusion of immature secretory granules (ISGs) gives rise to mature secretory granules (MSGs), the storage compartment in endocrine and neuroendocrine cells for hormones and neuropeptides. With the use of a cell-free fusion assay, we investigated which soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) molecules are involved in the homotypic fusion of ISGs. Interestingly, the SNARE molecules mediating the exocytosis of MSGs in neuroendocrine cells, syntaxin 1, SNAP-25, and VAMP2, were not involved in homotypic ISG fusion. Instead, we have identified syntaxin 6 as a component of the core machinery responsible for homotypic ISG fusion. Subcellular fractionation studies and indirect immunofluorescence microscopy show that syntaxin 6 is sorted away during the maturation of ISGs to MSGs. Although, syntaxin 6 on ISG membranes is associated with SNAP-25 and SNAP-29/GS32, we could not find evidence that these target (t)-SNARE molecules are involved in homotypic ISG fusion. Nor could we find any involvement for the vesicle (v)-SNARE VAMP4, which is known to be associated with syntaxin 6. Importantly, we have shown that homotypic fusion requires the function of syntaxin 6 on both donor as well as acceptor membranes, which suggests that t-t-SNARE interactions, either direct or indirect, may be required during fusion of ISG membranes.     

AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing "glue granules" that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.     

Site-specific cross-linking reveals a differential direct interaction of class 1, 2, and 3 ADP-ribosylation factors with adaptor protein complexes 1 and 3

We have used a site-specific photo-cross-linking approach to identify direct interactions between clathrin adaptor protein (AP)1 complexes and small GTPases of the ADP-ribosylation factor (ARF) family and to explore the specificity of this interaction on immature secretory granule (ISG) membranes. ISG membranes are a well-characterized, highly enriched preparation of membranes that has previously been shown to have the membrane-associated factors for ARF1 recruitment that are not present on artificial liposomes. All three classes of ARF proteins could be recruited to ISG membranes, displaying differential requirements for GTPgammaS. We found that ARF1, ARF5, and ARF6 interacted directly with the beta1-adaptin subunit of AP-1 in the presence of GTPgammaS. Furthermore, we observed a direct interaction between the switch 1 region of ARF1 and the N-terminal trunk domains of gamma- and beta1-adaptin. In addition, both ARF1 and ARF6 but not ARF5 interacted directly with the beta3- and delta-adaptin subunits of AP-3. No interaction was observed between AP-2 and any of the ARF proteins. Our results delineate the specificity and provide evidence of a direct interaction between different ARF proteins and the AP complexes AP-1 and AP-3 on natural ISG membranes and show that residues in the switch 1 region of ARF proteins can selectively bind to the trunk domains of these complexes.     

3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells

Since it was reported that components of immature secretory granules (ISGs) are different from those of mature secretory granules (MSGs) in rat parotid acinar cells, we have been considering that components of secretory granules (SGs) change dynamically during granule maturation. As the first step to understand the mechanism of granule maturation, we separated low-density detergent-resistant membrane fractions (DRMs) from purified SGs of rat parotid gland. When SGs were lysed by the detergent Brij-58, syntaxin6 and VAMP4 were found in DRMs that were different from the GM1a-rich DRMs containing VAMP2. Because syntaxin6 and VAMP4 are known to be related to granule formation, we attempted to separate DRMs from ISGs. To enrich for ISGs, glands were removed from rats 5h after intraperitoneal injection of isoproterenol and used to purify the newly synthesized granules. Compared to mature granules prepared without injection, these newly formed granules were lower in density and contained higher concentrations of syntaxin6, VAMP4, and gamma-adaptin. This composition is consistent with the characterizations of ISGs. DRMs isolated from the newly formed granules were GM1a-rich and contained syntaxin6, VAMP4, and VAMP2 together. Thus, our findings suggest that syntaxin6 and VAMP4 associate with a GM1a-rich membrane microdomain during granule formation but enter a separate membrane microdomain before transport from granules during maturation.     

Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA-mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.     

Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay

The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [³⁵S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process.     

An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

Clathrin provides an external scaffold to form small 50-100-nm transport vesicles. In contrast, formation of much larger dense-cored secretory granules is driven by selective aggregation of internal cargo at the trans-Golgi network; the only known role of clathrin in dense-cored secretory granules formation is to remove missorted proteins by small, coated vesicles during maturation of these spherical organelles. The formation of Weibel-Palade bodies (WPBs) is also cargo driven, but these are cigar-shaped organelles up to 5 mum long. We hypothesized that a cytoplasmic coat might be required to make these very different structures, and we found that new and forming WPBs are extensively, sometimes completely, coated. Overexpression of an AP-180 truncation mutant that prevents clathrin coat formation or reduced AP-1 expression by small interfering RNA both block WPB formation. We propose that, in contrast to other secretory granules, cargo aggregation alone is not sufficient to form immature WPBs and that an external scaffold that contains AP-1 and clathrin is essential.     

ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlF_n, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.     

Biogenesis of processing-competent secretory organelles in vitro.

Propeptide processing occurs in specific compartments of the secretory pathway, but how these processing-competent organelles are generated from their processing-incompetent precursor compartments is unknown. To dissect the process biochemically, we have developed a novel cell-free system reconstituting the production of processing-competent secretory granules in AtT-20 cells. Using donor membranes containing [(35)S]sulfate labeled pro-opiomelanocortin (POMC)(5) in the trans-Golgi, we can reconstitute cytosol- and ATP-dependent prohormone processing as well as incorporation of processed ACTH into immature secretory granules (ISGs). Under limiting cytosol conditions, both reactions are greatly stimulated by ADP-ribosylation factor 1 (ARF1) but not by the GDP-bound ARF1 T31N mutant. pH studies show that lumenal acidification, most likely due to ARF-mediated sorting of proton pumps and leaks during budding, confers processing competency to the resulting organelle. Surprisingly, comparison of onset of processing and ISG release reveals that they are distinct biochemical processes with different kinetics and separate cytosolic requirements. Moreover, ARF regulates the onset of prohormone processing but not ISG release. Our data suggest a two-step mechanism (onset of processing followed by ISG release) for the production of processing-competent organelles from the trans-Golgi and provide the first system with which these two steps may be individually dissected.     

Interactions between adaptor protein-1 of the clathrin coat and microtubules via type 1a microtubule-associated proteins.

The classical view suggests that adaptor proteins of the clathrin coat mediate the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into budding areas in the donor membrane. In the present study, we provide biochemical and morphological evidence that the adaptor protein 1 (AP-1) adaptor of the trans-Golgi network clathrin interacts with microtubules. AP-1 in cytosolic extracts interacted with in vitro assembled microtubules, and these interactions were inhibited by ATP depletion of the extracts or in the presence of 5'-adenylylimidodiphosphate. An overexpressed gamma-subunit of the AP-1 complex associated with microtubules, suggesting that this subunit may mediate the interaction of AP-1 with the cytoskeleton. Purified AP-1 did not interact with purified microtubules, but interaction occurred when an isolated microtubule-associated protein fraction was added to the reaction mix. The gamma-adaptin subunit of AP-1 specifically co-immunoprecipitated with a microtubule-associated protein of type 1a from rat brain cytosol. This suggests that type 1a microtubule-associated protein may mediate the association of AP-1 with microtubules in the cytoplasm. The microtubule binding activity of AP-1 was markedly inhibited in cytosol of mitotic cells. By means of its interaction with microtubule-associated proteins, we propose novel roles for AP-1 adaptors in modulating the dynamics of the cytoskeleton, the stability and shape of coated organelles, and the loading of nascent AP-1-coated vesicles onto appropriate microtubular tracks.     

100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties

The 100-110-kD proteins (alpha-, beta-, beta'-, and gamma-adaptins) of clathrin-coated vesicles and the 110-kD protein (beta-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), beta-COP was redistributed to the cytoplasm within 15 s, gamma-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the alpha-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect beta-COP binding or Golgi morphology but still induced redistribution of gamma-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of beta-COP and gamma-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release gamma-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which gamma-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTP gamma S, similar to the binding of beta-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.     

The trafficking of alpha 1-antitrypsin,a post-Golgi secretory pathway marker,in INS-1 pancreatic beta cells

A sulfated alpha1-antitrypsin (AAT), thought to be a default secretory pathway marker, is not stored in secretory granules when expressed in neuroendocrine PC12 cells. In search of a constitutive secretory pathway marker for pancreatic beta cells, we produced INS-1 cells stably expressing wild-type AAT. Because newly synthesized AAT arrives very rapidly in the Golgi complex, kinetics alone cannot resolve AAT release via distinct secretory pathways, although most AAT is secreted within a few hours and virtually none is stored in mature granules. Nevertheless, from pulse-chase analyses, a major fraction of newly synthesized AAT transiently exhibits secretogogue-stimulated exocytosis and localizes within immature secretory granules (ISGs). This trafficking occurs without detectable AAT polymerization or binding to lipid rafts. Remarkably, in a manner not requiring its glycans, all of the newly synthesized AAT is then removed from granules during their maturation, leading mostly to constitutive-like AAT secretion, whereas a smaller fraction (approximately 10%) goes on to lysosomes. Secretogogue-stimulated ISG exocytosis reroutes newly synthesized AAT directly into the medium and prevents its arrival in lysosomes. These data are most consistent with the idea that soluble AAT abundantly enters ISGs and then is efficiently relocated to the endosomal system, from which many molecules undergo constitutive-like secretion while a smaller fraction advances to lysosomes.     

Definition of the consensus motif recognized by gamma-adaptin ear domains

The heterotetrameric adaptor complex 1 (AP-1) and the monomeric Golgi-localized, gamma ear-containing, Arf-binding (GGA) proteins are components of clathrin coats associated with the trans-Golgi network and endosomes. The carboxyl-terminal ear domains (or gamma-adaptin ear (GAE) domains) of two gamma-adaptin subunit isoforms of AP-1 and of the GGAs are structurally similar and bind to a common set of accessory proteins. In this study, we have systematically defined a core tetrapeptide motif PsiG(P/D/E)(Psi/L/M) (where Psi is an aromatic residue), which is responsible for the interactions of accessory proteins with GAE domains. The definition of this motif has allowed us to identify novel GAE-binding partners named NECAP and aftiphilin, which also contain clathrin-binding motifs. These findings shed light on the mechanism of accessory protein recruitment to trans-Golgi network and endosomal clathrin coats.     

The structure and function of GGAs, the traffic controllers at the TGN sorting crossroads

GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding proteins) are a family of monomeric clathrin adaptor proteins that are conserved from yeasts to humans. Data published during the past four years have provided detailed pictures of the localization, domain organization and structure-function relationships of GGAs. GGAs possess four conserved functional domains, each of which interacts with cargo proteins including mannose 6-phosphate receptors, the small GTPase ARF, clathrin, or accessory proteins including Rabaptin-5 and gamma-synergin. Together with or independent of the adaptor protein complex AP-1, GGAs regulate selective transport of cargo proteins, such as mannose 6-phosphate receptors, from the trans-Golgi network to endosomes mediated by clathrin-coated vesicles.     

Biochemical dissection of AP-1 recruitment onto Golgi membranes

Recruitment of the Golgi-specific AP-1 adaptor complex onto Golgi membranes is thought to be a prerequisite for clathrin coat assembly on the TGN. We have used an in vitro assay to examine the translocation of cytosolic AP-1 onto purified Golgi membranes. Association of AP-1 with the membranes required GTP or GTP analogues and was inhibited by the fungal metabolite, brefeldin A. In the presence of GTP gamma S, binding of AP-1 to Golgi membranes was strictly dependent on the concentration of cytosol added to the assay. AP-1 recruitment was also found to be temperature dependent, and relatively rapid at 37 degrees C, following a lag period of 3 to 4 min. Using only an adaptor-enriched fraction from cytosol, purified myristoylated ARF1, and Golgi membranes, the GTP gamma S-dependent recruitment of AP-1 could be reconstituted. Our results show that the association of the AP-1 complex with Golgi membranes, like the coatomer complex, requires ARF, which accounts for the sensitivity of both to brefeldin A. In addition, they provide the basis for a model for the early biochemical events that lead to clathrin-coated vesicle formation on the TGN.     

ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma

Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.     

Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.