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.     

AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation

The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its beta1 and mu1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its beta1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated beta1 compared with phosphorylated mu1. Once on the membrane, the mu1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of mu1 (and mu2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70-mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.     

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.     

Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum.

Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expression further inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in untransformed and transformed NIH-3T3 cells. Adenoviral expression of caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to levels observed in untransformed NIH-3T3 cells. Cholesterol-rich detergent-resistant membrane domains or glycolipid rafts therefore invaginate independently of caveolin-1 expression to form endocytosis-competent caveolar vesicles via rapid dynamin-dependent detachment from the plasma membrane. Caveolin-1 stabilizes the plasma membrane association of caveolae and thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum.     

Structure and assembly of the endoplasmic reticulum. Biosynthetic sorting of endoplasmic reticulum proteins

We have studied the post-translational processing and the biosynthetic sorting of three protein components of murine endoplasmic reticulum (ER), ERp60, ERp72, and ERp99. In pulse-labeled MOPC-315 (where MOPC-315 represents mineral oil-induced plasmacytoma cells) plasmacytoma cells, no precursor forms of these proteins were detected and only ERp99 was sensitive to endoglycosidase H. The ERp99 oligosaccharide remained endoglycosidase H sensitive during a 3-h chase, and analysis by high performance liquid chromatography showed the predominant structure to be Man8GlcNAc2. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 plasmacytoma cells in order to directly study the biosynthetic sorting of both glycosylated and nonglycosylated ERps and have found no strong evidence to suggest these proteins ever leave the endoplasmic reticulum. In spite of their common sorting pathway, these proteins differ in their membrane orientation. Both ERp60 and ERp72 are entirely protected by the endoplasmic reticulum membrane while ERp99 appears to have a large domain exposed on the cytoplasmic face of the endoplasmic reticulum.     

Four secretory proteins synthesized by hepatocytes are transported from endoplasmic reticulum to Golgi complex at different rates

Pulse-chase experiments in conjunction with subcellular fractionation and quantitative immunoprecipitation have been used to study the intracellular transport of four secretory proteins, albumin, transferrin, prealbumin and retinol-binding protein, in isolated rat hepatocytes. The proteins were found to be transported from the endoplasmic reticulum (ER) to the Golgi complex (GC) at greatly different rates (t1/2 = 14-137 min), indicating that transport of secretory proteins between these organelles is effected by a selective, possibly receptor-mediated process and not through bulk phase transfers. The transport from the Golgi complex to the medium was rapid for all proteins (t1/2 approximately 15 min) and possibly occurred at the same rate. Consistent with these kinetic data, the amount of a rapidly transported protein (albumin) in the GC fraction was found to be high (relative to its amount in the ER fraction) whereas the amount of a slowly transported protein (transferrin) in the GC fraction was found to be low, as determined by radioimmunoassays.     

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.     

Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast

Genes that function in translocation of secretory protein precursors into the ER have been identified by a genetic selection for mutant yeast cells that fail to translocate a signal peptide-cytosolic enzyme hybrid protein. The new mutants, sec62 and sec63, are thermosensitive for growth and accumulate a variety of soluble secretory and vacuolar precursors whose electrophoretic mobilities coincide with those of the corresponding in vitro translated polypeptides. Proteolytic sensitivity of precursor molecules in extracts of mutant cells confirms that polypeptide translocation is blocked. Some form of interaction among the SEC61 (Deshaies, R. J., and R. Schekman. 1987. J. Cell Biol. 105:633-645), SEC62 and SEC63 gene products is suggested by the observation that haploid cells containing any pair of the mutations are inviable at 24 degrees C and show a marked enhancement of the translocation defect. The translocation defects of two mutants (sec62 and sec63) have been reproduced in vitro. sec63 microsomes display low and thermolabile translocation activity for prepro-alpha-factor (pp alpha F) synthesized with a cytosol fraction from wild type yeast. These gene products may constitute part of the polypeptide recognition or translocation apparatus of the ER membrane. Pulse-chase analysis of the translocation-defective mutants demonstrates that insertion of pp alpha F into the ER can proceed posttranslationally.     

Golgi-mediated vacuolar sorting of the endoplasmic reticulum chaperone BiP may play an active role in quality control within the secretory pathway

Quality control in the endoplasmic reticulum (ER) prevents the arrival of incorrectly or incompletely folded proteins at their final destinations and targets permanently misfolded proteins for degradation. Such proteins have a high affinity for the ER chaperone BiP and are finally degraded via retrograde translocation from the ER lumen back to the cytosol. This ER-associated protein degradation (ERAD) is currently thought to constitute the main disposal route, but there is growing evidence for a vacuolar role in quality control. We show that BiP is transported to the vacuole in a wortmannin-sensitive manner in tobacco (Nicotiana tabacum) and that it could play an active role in this second disposal route. ER export of BiP occurs via COPII-dependent transport to the Golgi apparatus, where it competes with other HDEL receptor ligands. When HDEL-mediated retrieval from the Golgi fails, BiP is transported to the lytic vacuole via multivesicular bodies, which represent the plant prevacuolar compartment. We also demonstrate that a subset of BiP-ligand complexes is destined to the vacuole and differs from those likely to be disposed of via the ERAD pathway. Vacuolar disposal could act in addition to ERAD to maximize the efficiency of quality control in the secretory pathway.     

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.     

KMS1 and KMS2, two plant endoplasmic reticulum proteins involved in the early secretory pathway

We have identified two endoplasmic reticulum (ER)-associated Arabidopsis proteins, KMS1 and KMS2, which are conserved among most species. Fluorescent protein fusions of KMS1 localised to the ER in plant cells, and over-expression induced the formation of a membrane structure, identified as ER whorls by electron microscopy. Hydrophobicity analysis suggested that KMS1 and KMS2 are integral membrane proteins bearing six transmembrane domains. Membrane protein topology was assessed by a redox-based topology assay (ReTA) with redox-sensitive GFP and confirmed by a protease protection assay. A major loop domain between transmembrane domains 2 and 3, plus the N- and C-termini were found on the cytosolic side of the ER. A C-terminal di(tri)-lysine motif is involved in retrieval of KMS1 and deletion led to a reduction of the GFP-KMS1 signal in the ER. Over-expression of KMS1/KMS2 truncations perturbed ER and Golgi morphology and similar effects were also seen when KMS1/KMS2 were knocked-down by RNA interference. Microscopy and biochemical experiments suggested that expression of KMS1/KMS2 truncations inhibited ER to Golgi protein transport.     

The binding of cyclic nucleotides to membranes of the endoplasmic reticulum of normal and neoplastic rat liver.

Studies are presented which demonstrate that the smooth and rough endoplasmic membranes of normal and neoplastic rat liver possess binding sites for cyclic nucleotides exhibiting a high degree of specificity. In contrast to normal liver, which has only a single binding site for cyclic AMP on membranes of the endoplasmic reticulum, cyclic AMP binding to the intracellular membranes of hepatoma 5123C and 7777 exhibits two apparent binding sites. The binding constant for cyclic AMP of one site on the tumor membranes is comparable to that of the normal liver, whereas the value of the second intrinsic association constant is 4- to 40-fold greater than liver. The possibility that the presence of the second cyclic AMP binding site might be a function of the rapid growth of the tumors was unlikely since membrane preparations from neonatal rats showed a single affinity association constant which was similar to that of normal liver. In addition, membranes of the endoplasmic reticulum of the Morris hepatomas 5123C and 7777 exhibit a binding site for cyclic GMP which is absent from the intracellular membranes of liver.     

Protein translocation into the endoplasmic reticulum: a light at the end of the tunnel

Intracellular transport of secretory of proteins and many membrane proteins in eukaryotic cells commences with their translocation into or across the membrane of the rough endoplasmic reticulum. Several components of the cellular machinery that mediates this process have been elucidated using in vitro assays or by genetic means. An analysis of how they function will depend on the ability to reassemble them into translocation-competent lipid vesicles.     

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.     

Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase Ero1-L alpha

Adiponectin is secreted from adipose tissue in response to metabolic effectors in order to sensitize the liver and muscle to insulin. Reduced circulating levels of adiponectin that usually accompany obesity contribute to the associated insulin resistance. The molecular mechanisms controlling the production of adiponectin are essentially unknown. In this report, we demonstrate that the endoplasmic reticulum (ER) oxidoreductase Ero1-L alpha and effectors modulating peroxisome proliferator-activated receptor gamma (PPAR gamma) and SIRT1 activities regulate secretion of adiponectin from 3T3-L1 adipocytes. Specifically, adiponectin secretion and Ero1-L alpha expression are induced during the early phase of adipogenesis but are then down-regulated during the terminal phase, coincident with an increased expression of SIRT1. Suppression of SIRT1 or activation of PPAR gamma enhances Ero1-L alpha expression and stimulates secretion of high-molecular-weight complexes of adiponectin in mature adipocytes. Suppression of Ero1-L alpha through expression of a corresponding small interfering RNA reduces adiponectin secretion during the differentiation of 3T3-L1 preadipocytes. Moreover, ectopic expression of Ero1-L alpha in Ero1-L alpha-deficient 3T3 fibroblasts stimulates the secretion of adiponectin following their conversion into adipocytes and prevents the suppression of adiponectin secretion in response to activation of SIRT1 by exposure to resveratrol. These findings provide a framework to understand the mechanisms by which adipocytes regulate secretion of adiponectin in response to various metabolic states.     

Tyrosine phosphorylation of S1PR1 leads to chaperone BiP-mediated import to the endoplasmic reticulum

Cell surface G protein–coupled receptors (GPCRs), upon agonist binding, undergo serine–threonine phosphorylation, leading to either receptor recycling or degradation. Here, we show a new fate of GPCRs, exemplified by ER retention of sphingosine-1-phosphate receptor 1 (S1PR1). We show that S1P phosphorylates S1PR1 on tyrosine residue Y¹⁴³, which is associated with recruitment of activated BiP from the ER into the cytosol. BiP then interacts with endocytosed Y¹⁴³-S1PR1 and delivers it into the ER. In contrast to WT-S1PR1, which is recycled and stabilizes the endothelial barrier, phosphomimicking S1PR1 (Y¹⁴³D-S1PR1) is retained by BiP in the ER and increases cytosolic Ca²⁺ and disrupts barrier function. Intriguingly, a proinflammatory, but non-GPCR agonist, TNF-α, also triggered barrier-disruptive signaling by promoting S1PR1 phosphorylation on Y¹⁴³ and its import into ER via BiP. BiP depletion restored Y¹⁴³D-S1PR1 expression on the endothelial cell surface and rescued canonical receptor functions. Findings identify Y¹⁴³-phosphorylated S1PR1 as a potential target for prevention of endothelial barrier breakdown under inflammatory conditions.     

Protein phosphorylation of the smooth and rough endoplasmic reticulum in normal and neoplastic liver of the rat.

Smooth and rough endoplasmic reticulum from rat liver and hepatomas exhibited endogenous protein kinase activity independent of adenosine 3':5'-monophosphate. The phosphorylation of smooth membranes by this process was consistently higher than that of rough membranes. When histone was added along with the smooth endoplasmic reticulum, cyclic AMP stimulated protein phosphorylation. Analysis of membrane-phosphorylated proteins by gel electrophoresis showed 5 major phosphorylated bands with estimated molecular weights of 155 000, 62 000, 50 000, 46 000 and 43 000, whereas major bands having estimated molecular weights of 62 000, 50 000 and 43 000 were found in membranes of the smooth endoplasmic reticulum of the Morris hepatoma 5123 C. Since previous studies in this and other laboratories have demonstrated the similarity of the protein components of membranes of the endoplasmic reticulum of normal liver and hepatoma, our findings indicate an inability of the protein kinase of hepatoma intracellular membranes to phosphorylate protein species that are found in membranes of both liver and the neoplasm.