Human luteinizing hormone and chorionic gonadotropin are targeted to a regulated secretory pathway in GH3 cells

LH is a dimeric glycoprotein hormone that is stored in the anterior pituitary and is released in response to GnRH, while the placental hormone, human CG (hCG), sharing the same alpha-subunit and a related beta-subunit, is secreted constitutively. In search of a determinant that allows sorting of LH into a regulated secretory pathway, the genes encoding the common alpha- and LH/CG beta-subunits were expressed in the GH3 rat pituitary tumor cell line, which contains a regulated secretory pathway. Steady state labeling and subsequent chase experiments showed that not only LH but also hCG can be sorted to a regulated secretory pathway; after an initial period of constitutive secretion, the mature forms of both hormones containing processed oligosaccharides were stored intracellularly, and their release was stimulated by either forskolin or KCl depolarization. In Chinese hamster ovary cells, which lack a regulated pathway and are devoid of storage granules, only hormones containing unprocessed N-linked oligosaccharides were found. In GH3 cells the LH beta-subunit was partially retained in an endoglycosidase H-sensitive form, presumably in the endoplasmic reticulum; the enzyme-resistant fraction was secreted through a regulated secretory pathway. A large fraction of the hCG beta-subunit was released constitutively, although some mature hCG beta-subunit accumulated in secretory granules and was released by forskolin. The common alpha-subunit was secreted constitutively with little intracellular accumulation of the mature forms. We conclude that the LH beta-subunit contains sufficient information to direct LH to a regulated pathway, and alpha:LH beta assembly is not a prerequisite for this targeting. The sorting of hCG to a regulated pathway in GH3 cells presumably reflects a structural similarity between LH and hCG. In addition, we have shown that GH3 cells can recognize the N-linked oligosaccharides on the gonadotropin subunits as substrates for sulfation.     

Procorticotrophin-releasing hormone: endoproteolytic processing and differential release of its derived peptides within AtT20 cells

Procorticotrophin-releasing hormone (proCRH) is expressed mainly in the hypothalamus and in the placenta, where it undergoes tissue-specific endoproteolysis. Our results show that within stably transfected AtT20/D16V cells proCRH is cleaved to generate two fragments of approximately 8 and 3 kDa which could account for proCRH(125-194) and proCRH(125-151), respectively, and a 4.5 kDa product which could account for mature IR-CRH(1-41). The immunofluorescence staining patterns for IR-CRH and IR-ACTH and their response of secretagogues indicate targeting of proCRH and POMC to the secretory pathway in transfected AtT20 cells. In this work, we have used a unique set of specific RIAs and IRMAs to the full length POMC and proCRH molecules and several products of endoproteolytic processing to assess if they could be released differentially in response to stimulation. Although the release of both IR-ACTH and IR-CRH peptides from transfected AtT20 cells is stimulated in response to exposure to high potassium stimulation (51 mM KCl/SmM CaCl2), the sorting index (SI) suggests that mature ACTH is sorted to the regulated secretory pathway 2.1-fold more efficiently than mature CRH(1-41). Mature ACTH is also sorted to the regulated secretory pathway 9-fold more efficiently than IR-proCRH(125-151). Also, mature CRH(1-41) is sorted to the regulated secretory pathway 3-fold more efficiently than IR-proCRH(125-151). These results therefore indicate that the intracellular mechanisms for the storage and release of POMC, proCRH and their endoproteolytic products differ and would sustain the hypothesis that within mammalian peptidergic cells, different biologically active peptides originating from the same or different precursor molecules, could be differentially released in response to specific stimuli. This would give these cells the capacity to finely regulate neurotransmitter release in response to environmental and physiological demands.     

Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events

PC2 and PC3, which is also known as PC1, are subtilisin-like proteases that are involved in the intracellular processing of prohormones and proneuropeptides. Both enzymes are synthesized as propolypeptides that undergo proteolytic maturation within the secretory pathway. An in vitro translation/translocation system from Xenopus egg extracts was used to investigate mechanisms in the maturation of pro-PC3 and pro-PC2. Pro-PC3 underwent rapid (t1/2 < 10 min) processing of the 88-kDa propolypeptide at the sequence RSKR83 to generate the 80-kDa active form of the enzyme. This processing was blocked when the active site aspartate was changed to asparagine, suggesting that an autocatalytic mechanism was involved. In this system, processing of pro-PC3 was optimal between pH 7.0 and 8.0 and was not dependent on additional calcium. These results are consistent with pro-PC3 maturation occurring at an early stage in the secretory pathway, possibly within the endoplasmic reticulum, where the pH would be close to neutral and the calcium concentration less than that observed in later compartments. Processing of pro-PC2 in the Xenopus egg extract was much slower than that of pro-PC3 (t1/2 = 8 h). It exhibited a pH optimum of 5.5-6.0 and was dependent on calcium (K0.5 = 2-4 mM). The enzymatic properties of pro-PC2 processing were similar to that of the mature enzyme. Further studies using mutant pro-PC2 constructs suggested that cleavage of pro-PC2 was catalyzed by the mature 68-kDa PC2 molecule. The results were consistent with pro-PC2 maturation occurring within a late compartment of the secretory pathway that contains a high calcium concentration and low pH.     

Cleavage of Alzheimer's amyloid precursor protein (APP) by secretases occurs after O-glycosylation of APP in the protein secretory pathway. Identification of intracellular compartments in which APP cleavage occurs without using toxic agents that interfere with protein metabolism

beta-Amyloid peptide (Abeta) is a principal component of parenchymal amyloid deposits in Alzheimer's disease. Abeta is derived from amyloid precursor protein (APP) by proteolytic cleavage. APP is subject to N- and O-glycosylation and potential tyrosine sulfation, following protein synthesis, and is then thought to be cleaved in an intracellular secretory pathway after or during these post-translational modifications. Studies utilizing agents that affect a series of steps in the protein secretory pathway have identified the possible intracellular sites of APP cleavage and Abeta generation within the protein secretory pathway. In the present study, using cells with normal protein metabolism, but expressing mutant APP with defective O-glycosylation, we demonstrated that the majority of APP cleavage by alpha-, beta-, and gamma-secretases occurs after O-glycosylation. Cells expressing the mutant APP noticeably decreased the generation of the intracellular APP carboxyl-terminal fragment (alphaAPPCOOH), a product of alpha-secretase, and both Abeta40 and Abeta42 in medium, a product of beta- and gamma-secretases. Furthermore, we found that the cells accumulate the mutant APP in intracellular reticular compartments such as the endoplasmic reticulum. Agents that could ambiguously affect the function of specific intracellular organelles and that may be toxic were not used. The present results indicate that APP is cleaved by alpha-, beta-, and gamma-secretases in step(s) during the transport of APP through Golgi complex, where O-glycosylation occurs, or in compartments subsequent to trans-Golgi of the APP secretory pathway.     

Digestibility and peptide patterns of modified lysozyme after hydrolyzing by protease

The metabolic pathway of Alzheimer's amyloid precursor protein (APP) involves restricted intracellular proteolysis by secretases, which leads to the secretion of the N-terminal soluble APP (sAPP) and the generation of a cell-associated C-terminal fragment. The precise cellular sites at which these processes occur remain unknown. In this report, we describe the route of APP sorting and the processing site using novel systems with and without sorting signals on the APP molecule. One system involves the replacement of the C-terminal ten amino acids of APP with Adenoviral E19 protein containing an endoplasmic reticulum (ER) retrieval signal (APPE19); the other involves deleting the last ten amino acids corresponding to the replaced site (APPdeltaC10). APPE19 localized mainly within the cis/medial Golgi compartment and exclusively suppresses the secretion of APP. In contrast, deletion of the C-terminal tail promotes sAPP secretion by a constitutive secretion pathway. Metabolic labeling followed by immunoprecipitation with anti-APP antibody revealed that APPE19 is rapidly degraded within 30 min and that the subsequent intracellular turnover rate is decreased with 40% of the protein retained within the cells even after a chase period a 3 h. In contrast, APPdeltaC10 is rapidly eliminated from the intracellular compartments and secreted into the culture medium. The surface internalization and recycling processes of this protein are relatively impaired compared with wild-type APP. The ratios of the levels of production to secretion of sAPP alpha, the N-terminal, soluble APP fragment released by alpha-secretase, are proportional to the secretion efficiencies among APP species, suggesting the localization of alpha-secretase within a compartment late in the constitutive secretion pathway. These secretion mutants which utilize ER targeting signals are useful tools for analyzing the location of secretases and the intracellular degradation system within a constitutive secretion pathway such as ER quality control.     

Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway

The CCC2 gene in the yeast Saccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p. Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease. In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway. First, like ccc2 mutants, sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p. Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p. These findings were sufficient to explain the respiratory deficiency of these mutants. Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization. Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway.     

Intracellular retention of the corticotrophin-releasing hormone (CRH) precursor within COS-7 cells

We investigated the intracellular localization of CRH in transiently transfected COS-7 cells expressing the full-length rat corticotropin-releasing hormone (CRH) precursor cDNA. CRH synthesized by transfected COS-7 cells is mainly stored intracellularly. In contrast, CHO-K1 cells expressing the same CRH precursor stored and released equal amounts of immunoreactive (IR)-CRH. Ultrastructural analysis revealed that CRH is stored in electron-dense aggregates in the RER of transiently transfected COS-7 cells and does not migrate into the Golgi apparatus. On the basis of the different intracellular localization, storage, and release of CRH in COS-7 and CHO-K1 cells, we hypothesize that the intracellular trafficking of CRH within the constitutive secretory pathway for protein secretion not only depends on its primary amino acid sequence but might also be influenced by intracellular conditions or factors.     

Clinical spectrum, therapeutic management, and follow-up of ventricular tachycardia in infants and young children

Prostaglandins mediate many biological processes. Arachidonic acid, the common precursor for all prostaglandins, is released from membrane phospholipids by both secretory and cytoplasmic forms of phospholipase A2. Free arachidonate is converted to prostaglandin H2, the common precursor to all prostanoids, by prostaglandin synthase. Both mitogen-induced prostaglandin synthesis in fibroblasts and endotoxin-induced prostaglandin synthesis in macrophages require expression of the inducible prostaglandin synthase-2; arachidonate released in these contexts is unavailable to prostaglandin synthase-1 constitutively present in fibroblasts or macrophages. In contrast to the results for fibroblasts and macrophages, prostaglandin synthesis by activated mast cells is mediated by prostaglandin synthase-1. Mast cell activation also provokes release of secretory phospholipase A2 (sPLA2). We now demonstrate that sPLA2 released from activated mast cells can mobilize arachidonate from distal Swiss 3T3 cells. This arachidonate is then used by prostaglandin synthase-1 present in 3T3 cells for prostaglandin synthesis. We thus distinguish two pathways for prostaglandin synthesis: (i) an intracellular pathway by which arachidonate released following ligand stimulation is made available only to prostaglandin synthase-2, and (ii) a transcellular pathway by which sPLA2 of proximal cells mobilizes, in distal cells, arachidonate available to prostaglandin synthase-1. Molecular and pharmacologic approaches to modulating prostaglandin-mediated events will differ for these two pathways.     

Targeting of tissue plasminogen activator into the regulated secretory pathway of neuroendocrine cells

Plasma levels of tissue plasminogen activator (tPA) increase rapidly in response to specific vasoactive agents, trauma, and neural stimulation. This response has been attributed to acute release of tPA from stored pools within the vascular endothelium and from catecholamine storage vesicles of chromaffin cells. We have tested directly whether tPA can be sorted into the regulated secretory pathway using the murine pituitary-derived neuroendocrine cell line AtT-20 transfected with tPA cDNA. Clones of AtT-20 cells expressing tPA were isolated, and targeting of tPA into the regulated secretory pathway was demonstrated by (1) stimulation of tPA secretion with 8-bromo-cAMP, the secretagogue which promotes the release of dense granule contents; (2) colocalization with ACTH, an endogenous protein that is stored in dense core granules; and (3) retention of newly synthesized tPA in the cell for prolonged periods of time. Laser scanning confocal microscopy analysis of cells immunostained with antibodies to tPA and ACTH showed colocalization at the tips of the neuritic processes under the cytoplasmic membrane, a region where dense granules are known to migrate after maturation. Treatment of the cells with 5 mM 8-bromo-cAMP for 30 min resulted in a 2.41+/-0.36-fold increase in tPA secretion. Both the magnitude of the stimulatory effect and the fraction of the intracellular tPA released were the same regardless of the tPA expression level in the various clones. Pulse-chase experiments showed that a portion of newly synthesized tPA is retained in the cell for at least 4 h and is released into the culture medium in response to 8-bromo-cAMP. These studies indicate that tPA, under the appropriate conditions, can be targeted into the regulated secretory pathway and can be stored for later release by cellular stimuli.     

The viral spike protein is not involved in the polarized sorting of coronaviruses in epithelial cells

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK(MHVR) cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK(MHVR) cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020-2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.     

The carboxy-terminal region of human lipoprotein lipase is necessary for its exit from the endoplasmic reticulum

Certain missense substitutions on the human lipase (hLPL) gene produce mutated proteins that are retained in different compartments along the secretory pathway. The purpose of the present study was to elucidate whether the C-terminal domain of the hLPL molecule could be important for secretion. We constructed by site-directed mutagenesis three carboxy-terminal mutated (F388-->Stop, K428-->Stop and K441-->Stop) hLPL cDNAs that were expressed in COS1 cells. Immunoblotting of cell extracts showed that all three constructs led to similar levels of protein. Both wild type (WT) hLPL and the truncated K441-->Stop hLPL were secreted to the extracellular medium, and presented a similar intracellular distribution pattern as shown by immunofluorescence. Neither F388-->Stop nor K428-->Stop hLPL protein was detected in cell medium. Immunofluorescence experiments showed that both truncated hLPL were retained within an intracellular compartment, which became larger. Double immunofluorescence analysis using antibodies against LPL and antiprotein disulfide isomerase as a marker showed that the truncated K428-->Stop hLPL was retained within the rough endoplasmic reticulum. This truncated protein was not found in other compartments in the secretory pathway, such as Golgi complex and lysosomes, indicating that it did not exit the endoplasmic reticulum. Further analysis of the C-terminal region of the LPL molecular model showed both that F388-->Stop and K428-->Stop hLPL truncated proteins are highly hydrophobic. As retention of secretory proteins in the rough endoplasmic reticulum is a quality control mechanism of the secretory pathway, we conclude that the C-terminal domain of hLPL is critical for correct intracellular processing of the newly synthesized protein.     

The occurrence of protein kinase C theta and lambda isoforms in retina of different species

Salmonella species are intracellular facultative pathogens which survive within phagocytic cells such as macrophages and proliferate inside vacuoles of epithelial cells. Early reports suggested that the capacity for surviving within macrophages was due to the inhibitory effect on the phagosome-lysosome fusion event induced by intracellular Salmonella. However, recent cell biology data, obtained both with phagocytic and epithelial cells, have shown that Salmonella-containing phagosomes have large amounts of lysosomal membrane glycoproteins (lgp), major components of the lysosomal membrane. This apparent discrepancy has partly been clarified at least in epithelial cells: the Salmonella-containing phagosome fuses with lgp-rich compartment different from the classical mature lysosome, as they do not contain certain lysosomal enzymes and are not connected with the endocytic route. Therefore, Salmonella seems to use an alternative strategy not merely based on the inhibition of phagosome-lysosome fusion event. This strategy essentially involves acquisition of only certain lysosomal components to form a specialized phagosomal compartment in which to survive or proliferate intracellularly. These observations have also exemplified the potential use of intracellular bacterial pathogens as biological probes to understand normal biological aspects of the eukaryotic cell. The intracellular lifestyle of Salmonella will undoubtedly provide new insights into the process of lysosome biogenesis.     

Protein secretion: puzzling receptors

All known sorting receptors for soluble cargo in the secretory pathway are transmembrane proteins. For sorting to the regulated pathway, however, a subpopulation of secretory proteins, associated with the membrane but not membrane-spanning, appears to link cargo and membrane in storage granule biogenesis.     

Intracellular storage and regulated plasma membrane expression of human complement receptor type 1 in rat basophil leukemia cell transfectants

Polymorphonuclear neutrophils (PMN) contain multiple distinct secretory compartments that are sequentially mobilized during cell activation. Complement receptor type 1 (CR1) is a marker for a readily mobilizable secretory vesicle compartment, which can undergo exocytic fusion with the plasma membrane independently of secretion of traditional granule contents. The basis for the formation of these distinct compartments is incompletely understood. Primary and secondary granules are generated directly from the Golgi complex during different stages of development of the cell, obviating the need for sorting signals for proper packaging of their constituents. To determine whether the secretory vesicles are formed in a similar manner, we studied a stable rat basophilic leukemia cell line (RBL-CR1) transfected with a plasmid containing the cDNA of human CR1 driven by a viral promoter. The CR1 was present primarily intracellularly in small vesicles resembling the CR1 storage pools in resting PMN. Activation of RBL-CR1 resulted in translocation of intracellular CR1 to the plasma membrane, with mobilization requirements different from those of the classical RBL granules. Thus, in RBL-CR1, continuously synthesized CR1 is stored and upregulated in much the same way as in PMN. This suggests that differential timing of gene expression is not essential for proper storage of CR1 and that other sorting mechanisms are involved, which can be studied in RBL-transfectants.     

Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in cpefat mice associated with a carboxypeptidase E mutation

Cpefat mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia-diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in Cpefat mice. In Cpefat mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high K+ stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of Cpefat mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of Cpefat mice.     

Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast

Sorting of membrane proteins between compartments of the secretory pathway is mediated in part by their transmembrane domains (TMDs). In animal cells, TMD length is a major factor in Golgi retention. In yeast, the role of TMD signals is less clear; it has been proposed that membrane proteins travel by default to the vacuole, and are prevented from doing so by cytoplasmic signals. We have investigated the targeting of the yeast endoplasmic reticulum (ER) t-SNARE Ufe1p. We show that the amino acid sequence of the Ufe1p TMD is important for both function and ER targeting, and that the requirements for each are distinct. Targeting is independent of Rer1p, the only candidate sorting receptor for TMD sequences currently known. Lengthening the Ufe1p TMD allows transport along the secretory pathway to the vacuole or plasma membrane. The choice between these destinations is determined by the length and composition of the TMD, but not by its precise sequence. A longer TMD is required to reach the plasma membrane in yeast than in animal cells, and shorter TMDs direct proteins to the vacuole. TMD-based sorting is therefore a general feature of the yeast secretory pathway, but occurs by different mechanisms at different points.     

A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3gamma, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA-DM and the invariant chain (I chain) complexed to HLA-DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR-I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR-I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR-I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR-I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR-I chain until their intended rendezvous in late endocytic compartments.     

Multiresidue method for determination of organophosphorus pesticides in vegetables

Yeasts combine the advantages of fast and easy handling with the potential to perform eukaryotic post-translational modifications and are for this reason interesting hosts for heterologous production of G-protein-coupled receptors. The possibility to connect foreign receptors to a yeast internal MAP kinase pathway was used to establish yeast-based systems for high-throughput screening of compound libraries. In addition, yeasts have the potential for high level production of G-protein-coupled receptors. In this field, non-Saccharomyces yeasts seems to be interesting alternatives to S. cerevisiae, as well as to systems based on higher eukaryotic cells.     

CCK biosynthesis and processing: recent progress and future challenges

The peptide cholecystokinin (CCK), like other peptides which pass through the regulated secretory pathway, undergoes a number of post-translational modifications during its biosynthesis including tyrosine sulfation, endoproteolytic cleavage, and trimming by carboxypeptidases. This minireview summarizes what is known about this process in endocrine cells and in the Cpe(fat)/Cpe(fat) mouse and points out what challenges remain for future research.     

A dileucine-like sorting signal directs transport into an AP-3-dependent, clathrin-independent pathway to the yeast vacuole

Transport of yeast alkaline phosphatase (ALP) to the vacuole depends on the clathrin adaptor-like complex AP-3, but does not depend on proteins necessary for transport through pre-vacuolar endosomes. We have identified ALP sequences that direct sorting into the AP-3-dependent pathway using chimeric proteins containing residues from the ALP cytoplasmic domain fused to sequences from a Golgi-localized membrane protein, guanosine diphosphatase (GDPase). The full-length ALP cytoplasmic domain, or ALP amino acids 1-16 separated from the transmembrane domain by a spacer, directed GDPase chimeric proteins from the Golgi complex to the vacuole via the AP-3 pathway. Mutation of residues Leu13 and Val14 within the ALP cytoplasmic domain prevented AP-3-dependent vacuolar transport of both chimeric proteins and full-length ALP. This Leucine-Valine (LV)-based sorting signal targeted chimeric proteins and native ALP to the vacuole in cells lacking clathrin function. These results identify an LV-based sorting signal in the ALP cytoplasmic domain that directs transport into a clathrin-independent, AP-3-dependent pathway to the vacuole. The similarity of the ALP sorting signal to mammalian dileucine sorting motifs, and the evolutionary conservation of AP-3 subunits, suggests that dileucine-like signals constitute a core element for AP-3-dependent transport to lysosomal compartments in all eukaryotic cells.