AP-3 adaptor functions in targeting P-selectin to secretory granules in endothelial cells

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro . The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet α-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes.     

The role of dibasic residues in prohormone sorting to the regulated secretory pathway. A study with proneurotensin

The mechanisms by which prohormone precursors are sorted to the regulated secretory pathway in neuroendocrine cells remain poorly understood. Here, we investigated the presence of sorting signal(s) in proneurotensin/neuromedin N. The precursor sequence starts with a long N-terminal domain followed by a Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin)-Lys-Arg- sequence and a short C-terminal tail. An additional Arg-Arg dibasic is contained within the neurotensin sequence. Mutated precursors were expressed in endocrine insulinoma cells and analyzed for their regulated secretion. Deletion mutants revealed that the N-terminal domain and the Lys-Arg-(C-terminal tail) sequence were not critical for precursor sorting to secretory granules. In contrast, the Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin) sequence contained essential sorting information. Point mutation of all three dibasic sites within this sequence abolished regulated secretion. However, keeping intact any one of the three dibasic sequences was sufficient to maintain regulated secretion. Finally, fusing the dibasic-containing C-terminal domain of the precursor to the C terminus of beta-lactamase, a bacterial enzyme that is constitutively secreted when expressed in neuroendocrine cells, resulted in efficient sorting of the fusion protein to secretory granules in insulinoma cells. We conclude that dibasic motifs within the neuropeptide domain of proneurotensin/neuromedin N constitute a necessary and sufficient signal for sorting proteins to the regulated secretory pathway.     

Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway

The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.     

Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.     

Sorting of an exocrine secretory protein to the regulated secretory pathway in endocrine cells

The effect of exogenous polyamines on electrolyte leakage, chilling index, polygalacturonase activity (PG), ethylene production, and firmness in zucchini squash fruits stored for 12 days at 2 degrees C or 10 degrees C, 85-90% RH was evaluated. Fruits were infiltrated with putrescine (PUT) spermidine (SPD) and spermine (SPM) at 0.1, 0.25, 0.5, 2.0, and 4.0 mM. All polyamines exerted a protective effect on cell and organelle membranes. The most effective was SPD, which reduced electrolyte leakage between 62% and 82%, compared to control fruits stored at 2 degrees C. At 10 degrees C they did not exhibit chilling injury (CI) symptoms, while at 2 degrees C SPM (0.5 mM) and SPD (0.5 mM) diminished them 92% and 100%, respectively; which extended storage life for 8-10 days at 2 degrees C. High concentrations of polyamines (>2.0 mM) caused the appearance of CI symptoms. PG activity diminished proportionally to the concentration of polyamine except for the concentration at 4.0 mM. No significant changes were observed in ethylene production.     

COPI-mediated protein and lipid sorting in the early secretory pathway

Summary The major function of the secretory pathway of eukaryotes is to maintain the compartmental organization of the endomembrane system and organelle-associated functions by proper distribution of newly synthesized molecules. Protein and lipid transport is mediated by vesicular intermediates that connect the various organelles throughout this pathway. This principle enables the eukaryotic cell to actively sort proteins and lipids at every level of this route, in both the anterograde and the retrograde direction. Here, we discuss the molecular mechanisms of nonclathrin (COPI and COPII)-coated-vesicle biogenesis and how transport vesicle formation is linked to protein and lipid sorting in the early secretory pathway.     

Basic mechanisms of secretion: sorting into the regulated secretory pathway.

Targeting proteins to their correct cellular location is crucial for their biological function. In neuroendocrine cells, proteins can be secreted by either the constitutive or the regulated secretory pathways but the mechanism(s) whereby proteins are sorted into either pathway is unclear. In this review we discuss the possibility that sorting is either an active process occurring at the level of the trans-Golgi network, or that sorting occurs passively in the immature granules, The possible involvement of protein-lipid interactions in the sorting process is also raised.     

An essential role for a protein and lipid kinase complex in secretory protein sorting

Yeast genetics has identified more than 40 genes involved in the biogenesis and maintenance of the yeast lysosome-like vacuole. Recent data on two of these genes, VPS15 and VPS34, are beginning to provide some fundamental insights into the mechanisms governing protein sorting within the eukaryotic secretory pathway. VPS15 and VPS34 encode a novel protein kinase and a phosphatidylinositol 3-kinase, respectively, that function together as components of a membrane-associated signal transduction complex. These studies of the VPS15-VPS34 complex indicate that intracellular protein trafficking decisions may be regulated by protein phosphorylation and phosphatidylinositol signalling events.     

Dibasic cleavage site is required for sorting to the regulated secretory pathway for both pro- and neuropeptide Y

To investigate the signals governing routing of biologically active peptides to the regulated secretory pathway, we have expressed mutated and non-mutated proneuropeptide Y (ProNPY) in pituitary-derived AtT20 cells. The mutations were carried out on dibasic cleavage site and or ProNPY C-terminal sequence. Targeting to the regulated secretory pathway was studied using protein kinase A (8-BrcAMP), protein kinase C (phorbol myristate acetate) specific activators and protein synthesis inhibitor cycloheximide, and by pulse chase. The analysis of expressed peptides in cells and culture media indicated that: neuropeptide Y (NPY) and ProNPY were differently secreted, whilst NPY was exclusively secreted via regulatory pathway; ProNPY was secreted via regulated and constitutive-like secretory pathways. ProNPY secretion behaviour was not Proteolytic cleavage efficiency-dependent. The dibasic cleavage was essential for ProNPY and NPY cAMP-dependent regulated secretion and may have function as a retention signal.     

Lectin control of protein folding and sorting in the secretory pathway

Glycan moieties are essential for folding, sorting and targeting of glycoproteins through the secretory pathway to various cellular compartments. The molecular mechanisms that underlie these processes, however, are only now coming to light. Recent crystallographic and NMR studies of proteins located in the endoplasmic reticulum (ER), Golgi complex and ER-Golgi intermediate compartment have illuminated their roles in glycoprotein folding and secretion. Calnexin and calreticulin, both ER-resident proteins, have lectin domains that are crucial for their function as chaperones. The crystal structure of the carbohydrate-recognition domain of ER-Golgi intermediate compartment (ERGIC)-53 complements the biochemical and functional characterization of the protein, confirming that a lectin domain is essential for the role of this protein in sorting and transfer of glycoproteins from the ER to the Golgi complex. The lectin domains of calnexin and ERGIC-53 are structurally similar, although there is little primary sequence similarity. By contrast, sequence similarity between ERGIC-53 and vesicular integral membrane protein (VIP36), a Golgi-resident protein, leaves little doubt that a similar lectin domain is central to the transport and/or sorting functions of VIP36. The theme emerging from these studies is that carbohydrate recognition and modification are central to mediation of glycoprotein folding and secretion.     

Insertion of dibasic residues directs a constitutive protein to the regulated secretory pathway.

The mechanisms for sorting proteins to the regulated secretory pathway (RSP) remains poorly understood. We recently reported that dibasic sequences that are cleaved by pro-protein convertases (PCs) in pro-neurotensin also acted as sorting signal for the precursor. Here we addressed two questions regarding the role of dibasics as sorting signal: (i) Are dibasics sufficient to direct proteins to the RSP? (ii) Do they sort proteins by virtue of their interaction with PCs? The first question was studied by inserting dibasics in beta-lactamase, a constitutively secreted protein and comparing the regulated secretion of beta-lactamase to that of its mutant in transfected endocrine cells. The second question was investigated by comparing the regulated release of pro-neurotensin in PC12 cells that are devoid of PCs to that in PC1- and PC2-transfected PC12 cells. The data show that the mutant beta-lactamase was indeed targeted in part to the RSP and that pro-neurotensin was sorted to the RSP without the assistance of the PCs, thus indicating that dibasics can act as sorting signal by themselves independently of their interaction with PCs.     

A conserved alpha-helix at the amino terminus of prosomatostatin serves as a sorting signal for the regulated secretory pathway

Mammalian prosomatostatin (PSST) contains the bioactive peptides SST-14 and SST-28 at the COOH-terminal end of the molecule and a putative sorting signal in the propeptide segment for targeting the precursor to the regulated secretory pathway. The NH(2)-terminal segment of PSST consists of an amphipathic alpha-helix, which has been totally conserved throughout vertebrate evolution. We have analyzed the PSST-(3--15) region for sorting function by alanine scanning and deletional mutagenesis. Mutants created were stably expressed in AtT-20 cells. Regulated secretion was studied by analyzing basal and stimulated release of SST-14 LI and by immunocytochemistry for staining of SST-14 LI in punctate granules. Deletion of the PSST-(3--15) segment blocked regulated secretion and rerouted PSST for constitutive secretion as unprocessed precursor. Alanine scanning mutagenesis identified the region Pro(5)--Gln(12) as being important in precursor targeting, with Leu(7) and Leu(11) being critical. Molecular modeling demonstrated that these two residues are located in close proximity on a hydrophobic surface of the alpha-helix. Disruption of the alpha-helix did not impair the ability of PSST to be processed at the COOH terminus to SST-14 and SST-28. Processing, however, was shifted to the early compartments of the secretory pathway rather than storage granules and was relatively inefficient.     

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.     

Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.     

The AP-1 adaptor complex binds to immature secretory granules from PC12 cells, and is regulated by ADP-ribosylation factor

Immature secretory granules (ISGs) in endocrine and neuroendocrine cells have been shown by morphological techniques to be partially clathrin coated (Orci, L., M. Ravazzola, M. Amherdt, D. Lonvard, A. Perrelet. 1985a. Proc. Natl. Acad. Sci. USA. 82:5385-5389; Tooze, J., and S. A. Tooze. 1986. J. Cell Biol. 103:839-850). The function, and composition, of this clathrin coat has remained an enigma. Here we demonstrate using three independent techniques that immature secretory granules isolated from the rat neuroendocrine cell line PC12 have clathrin coat components associated with their membrane. To study the nature of the coat association we have developed an assay whereby the binding of the AP-1 subunit gamma-adaptin to ISGs was reconstituted by addition of rat or bovine brain cytosol. The amount of gamma-adaptin bound to the ISGs was ATP independent and was increased fourfold by the addition of GTPgammaS. The level of exogenous gamma-adaptin recruited to the ISG was similar to the level of gamma-adaptin present on the ISG after isolation. Addition of myristoylated ARF1 peptide stimulated binding. Reconstitution of the assay using AP-1 adaptor complex and recombinant ARF1 provided further evidence that ARF is involved in gamma-adaptin binding to ISGs; BFA inhibited this binding. Trypsin treatment and Trisstripping of the ISGs suggest that additional soluble and membrane-associated components are required for gamma-adaptin binding.     

The C-terminus of prohormone convertase 2 is sufficient and necessary for Raft association and sorting to the regulated secretory pathway

Prohormone convertase 2 (PC2) is a member of the subtilisin family of proteases involved in prohormone maturation in the granules of the regulated secretory pathway (RSP). It has been suggested that targeting of this enzyme to the RSP is dependent on its association with lipid rafts in membranes at the trans-Golgi network. Here, we investigate the orientation of PC2 in granule membranes and the role of the C-terminus in sorting of the enzyme to the RSP. Molecular modeling and circular dichroism showed that this domain of PC2 forms an alpha-helix and inserts into artificial membranes. Furthermore, we show that the C-terminus of PC2 can be biotinylated at the C-terminus in intact chromaffin granules, indicating that it is a transmembrane protein. To determine if the PC2 C-terminus is necessary for raft association and sorting, we transfected a chimera of CPEDelta15 (carboxypeptidase E without the last 15 residues) and the last 25 residues of PC2 (CPEDelta15-PC2), and a truncated PC2 mutant with the last 6 residues deleted (PC2Delta6) into Neuro2a cells. Whereas CPEDelta15 was not raft-associated or sorted to the RSP, addition of the 25 residues of PC2 C-terminus to CPEDelta15 restored raft association and localization to the RSP granules, as determined by immunocytochemistry. Deletion of the last 6 residues of PC2 eliminated lipid raft association and sorting of PC2Delta6 to the RSP. These results showed that the PC2 C-terminus confers raft association and is sufficient and necessary for sorting PC2 to the RSP.     

Sorting of the neuroendocrine secretory protein Secretogranin II into the regulated secretory pathway: role of N- and C-terminal alpha-helical domains

Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative alpha-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane.     

Identification of ER proteins involved in the functional organisation of the early secretory pathway in Drosophila cells by a targeted RNAi screen

Background

In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation.

Results

To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER.

Conclusions

This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation.     

Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway.

P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.