Granule-associated Proteins Research Articles

Identification of Routing Determinants in the Cytosolic Domain of a Secretory Granule-associated Integral Membrane Protein

We have investigated the trafficking of integral membrane peptidylglycine alpha-amidating monooxygenase (PAM) in the neuroendocrine AtT-20 cell line. This bifunctional enzyme has two domains which together catalyze the COOH-terminal alpha-amidation of peptidylglycine substrates yielding amidated products stored in secretory granules. As soluble proteins, both catalytic domains were independently targeted to secretory granules. In contrast, membrane PAM was largely localized to the trans-Golgi network (TGN). Upon truncation of its cytoplasmic COOH-terminal domain, membrane PAM was less efficiently cleaved by secretory granule enzymes and accumulated on the plasma membrane. When transferred to the lumenal domain of the interleukin 2 receptor alpha-chain (Tac protein), the cytoplasmic domain of PAM caused rerouting of Tac from the surface to the TGN and supported internalization of Tac antibody from the plasma membrane. To define sequences in the cytoplasmic domain of integral membrane PAM involved in its trafficking, we expressed PAM proteins containing truncations, deletions, or point mutations in the COOH-terminal cytoplasmic domain. PAM proteins were not retained in the TGN when half of the cytoplasmic domain was deleted; such proteins accumulated on the plasma membrane, were not efficiently internalized, and were cleaved to generate a bifunctional PAM protein that was not stored in secretory granules. A tyrosine-based internalization motif was identified, which was not required for efficient cleavage of full-length integral membrane PAM by secretory granule enzymes. Deletion of an 18-amino acid domain surrounding this Tyr residue both diminished cleavage of membrane PAM by secretory granule enzymes and eliminated internalization of PAM from the plasma membrane. The cytoplasmic domain is responsible for retaining membrane PAM in the TGN and for retrieving membrane PAM from the cell surface, while the lumenal catalytic domains of PAM appear to be responsible for targeting the protein to secretory granules.

Dopaminergic regulation of secretory granule-associated proteins in rat intermediate pituitary.

The biosynthesis of peptides requires the synthesis of the prohormone, several biosynthetic processing enzymes, and other granule constituents. We have investigated the regulated expression of proopiomelanocortin (POMC) and five enzymes essential for the processing of POMC to smaller, bioactive peptides in intermediate pituitary melanotropes. Rats were treated with a dopaminergic agonist (bromocriptine) or antagonist (haloperidol) for periods ranging from 1 h to 5 days, followed by analyses of mRNA levels and protein biosynthetic rates. Multiplex RNase protection assays showed that bromocriptine treatment caused a striking decrease in POMC mRNA levels, and significant decreases in mRNA levels for prohormone convertase 2 (PC2), carboxypeptidase H (CPH), and peptidylglycine alpha-amidating monooxygenase (PAM). Smaller increases in mRNA levels were seen after haloperidol stimulation. Protein biosynthetic rates changed more profoundly than mRNA levels at short drug treatment times, indicating a role for translational effects after treatment with bromocriptine and with haloperidol. The homogeneous population of melanotropes in the intermediate lobe of the pituitary allows a quantitative analysis of transcript levels and biosynthetic rates. POMC mRNA levels are 200-1,000-fold higher than levels of any of the processing enzyme mRNAs, and POMC biosynthetic rates exceed those of PC2, PC1, and PAM by 1,000-10,000-fold.

New knowledge about the PHA-locus and P(3HB) granule-associated proteins in Chromatium vinosum

The isolation of poly(3-hydroxybutyric acid) granules of Chromatium vinosum D was re-examined. Beside the PHA synthase and a 17 kDa protein, a 14 kDa protein was identified as predominant granule-associated protein. The M r as well as the N-terminal amino acid sequence exhibited identity to ORF5Cv, which is located within the pha-locus of C. vinosum. In addition, sequence alignements revealed new information about ORF4Cv, which is also located within the pha-locus, and about the 17 kDa protein, which exhibited homology to heat shock proteins recently detected in Escherichia coli.

Trimeric G Proteins Control Regulated Exocytosis in Bovine Chromaffin Cells: Sequential Involvement of Go Associated With Secretory Granules and Gi3 Bound to the Plasma Membrane

Regulated secretion requires both calcium and MgATP. Studies in diverse secretory systems indicate that ATP is required to prime the exocytotic apparatus whereas Ca2+ triggers the final ATP-independent fusion event. In this paper, we examine the possible role of trimeric G proteins in these two steps of exocytosis in chromaffin cells. We show that in the presence of low concentrations of Mg2+, mastoparan selectively stimulates G proteins associated with purified chromaffin granule membranes. Under similar conditions in permeabilized chromaffin cells, mastoparan inhibits ATP-dependent secretion but is unable to trigger ATP-independent release. This inhibitory effect of mastoparan on secretion was specifically reversed by anti-Galphao antibodies and a synthetic peptide corresponding to the carboxyl terminus of Galphao. In contrast, mastoparan required millimolar Mg2+ for the activation of plasma membrane-bound G proteins and stimulation of ATP-independent secretion in permeabilized chromaffin cells. The latter effect was completely inhibited by anti-Galphai3. By confocal immunofluorescence and immunoreplica analysis, we provide evidence that in chromaffin cells Go is preferentially associated with secretory granules, while Gi3 is essentially present on the plasma membrane. Our findings suggest that these two trimeric G proteins act in series in the exocytotic pathway in chromaffin cells: a secretory granule-associated Go protein controls the ATP-dependent priming reaction, whereas a plasma membrane-bound Gi3 protein is involved in the late calcium-dependent fusion step, which does not require ATP.

Occurrence of polyhydroxyalkanoic acid granule-associated proteins related to the Alcaligenes eutrophus H16 GA24 protein in other bacteria.

Fifty different polyhydroxyalkanoic acid (PHA)-accumulating bacterial strains were investigated for the occurrence of phasin proteins bound to PHA granules and related to the GA24 protein of Alcaligenes eutrophus H16, by isolating PHA granules and Western blot analysis of granule-associated proteins employing antibodies raised against the GA24 protein. It could be demonstrated that th PHA granules of many poly(3-hydroxybutyrate)-accumulating bacteria exhibited a similar protein pattern, and a predominant protein of 24 +/- 2 kDa occurred in the granules of A. eutrophus strains A7, CH34, JMP222, N9A and TF93 exhibiting N-terminal amino acid sequences identical to that of the GA24 protein. Proteins bound to the granules of A. latus, Burkholderia caryophyli, B. cepacia, B. solanacearum, Pseudomonas glathei, Rhodobacter sphaeroides and Telluria mixta also gave positive immunoreactions. Granule-associated proteins of small size also occurred in various strains of the Gram positive bacteria Bacillus megaterium and R. ruber as well as in the Gram-negative bacteria Azotobacter sp., Chromatium vinosum, Comamonas acidovorans, Methylobacterium sp., Mycoplana rubra, Paracoccus denitrificans, Pseudomonas sp., Rhodospirillum rubrum, Rubrivivax gelatinosus and Thiocystis violacea; however, they gave no immunoreaction. This study clearly demonstrated that phasins are wide-spread if not essential in PHA-accumulating bacteria.

Occurrence of polyhydroxyalkanoic acid granule-associated proteins related to the Alcaligenes eutrophus H16 GA24 protein in other bacteria

Fifty different polyhydroxyalkanoic acid (PHA)-accumulating bacterial strains were investigated for the occurrence of phasin proteins bound to PHA granules and related to the GA24 protein of Alcaligenes eutrophus H16, by isolating PHA granules and Western blot analysis of granule-associated proteins employing antibodies raised against the GA24 protein. It could be demonstrated that the PHA granules of many poly(3-hydroxybutyrate)-accumulating bacteria exhibited ja similar protein pattern, and a predominant protein of 24 ± 2 kDa occurred in the granules of A. eutrophus strains A7, CH34, JMP222, N9A and TF93 exhibiting N-terminal amino acid sequences identical to that of the GA24 protein. Proteins bound to the granules of A. latus, Burkholderia caryophvli B. cepacia B. solanacearum, Pseudomonas glathei. Rhodobacter sphaeroides and Telluria mixta also gave positive immunoreactions. Granule-associated proteins of small size also; occurred in various strains of the Gram-positive bacteria Bacillus megaterium and R. ruher as well as in the Gram-negative bacteria Azotohacter sp., Chromatium vinosum, Comamonas acidovorans, Methylobacterium sp., Mycoplana ruhra, Paracoccus denitrificans, Pseudomonas sp., Rhodospirillum ruhrum, Rubrivivax gelatinosus and Thiocystis violacea; however, they gave no immunoreaction. This study clearly demonstrated that phasins are wide-spread if not essential in PHA-accümulating bacteria.

Regulation of azurophil granule-associated serine protease genes during myelopoiesis.

During myelopoiesis, the primary or azurophil granules are synthesized at the promyelocyte stage of development. The biogenesis of these granules requires the coordinated regulation of a large number of genes that comprise the constituent proteins of the granules themselves. The genes encoding the granule-associated proteins appear to be regulated primarily at the level of gene transcription; that is, most of the granule genes appear to be transcriptionally activated at the beginning of the promyelocyte stage and are transcriptionally repressed at the transition to myelocytes. The cis-acting DNA elements and trans-regulatory factors that control the regulation of granule genes are just beginning to be defined. The granule-associated genes are dispersed throughout the genome; however, several of the serine protease genes expressed in these granules are tightly clustered and the tight clustering may play an important role for their regulation. Recent transgenic studies suggest that DNA elements required for myeloid-specific targeting may be located just upstream from or very near to the granule genes themselves. DNA elements similar to the globin locus control region will probably play a role in the expression of these genes, although locus control elements have not yet been defined for any azurophil granule genes. Future studies directed at defining these key regulatory elements will enhance our understanding of the molecular events that underlie myeloid development.

Cytolytic effector mechanisms of human CD4 + cytotoxic T lymphocytes

To elucidate mechanisms by which human CD4 + cells mediated cytolytic activity, we studied the expression of cytolytic proteins and the effects of inhibitors and mAbs on T-cell clones. Of seven cytolytic CD4 + clones, three were specific for the HLA-DR17, while four recognized DR18. Anti-HLA-DR mAb and anti-CD4 mAb blocked lysis. In addition, N α - p-tosyl- l-lysine chloromethylketone (TLCK), a serine esterase inhibitor, as well as cytochalasin B and monensin, antagonists of secretory pathways, inhibited CD4 + CTLs, whereas the absence of extracellular Ca ++ or the presence of Ca ++ channel blockers partially inhibited cytotoxicity. CD4 + CTLs induced apoptosis of target cell nuclei and membrane damage simultaneously. The CD4 + clones synthesized petforin and granzyme B and expressed the granule-associated protein TIA-1. Our studies indicate that two distinct mechanisms may contribute to cytolysis by CD4 + clones: (1) a Ca ++-dependent mechanism associated with the cytotoxic granules and (2) a Ca ++-insensitive mechanism.

Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions.

Some mathematical calculations were done that provided information about the structure and biochemistry of polyhydroxyalkanoic acid (PHA) granules and about the amounts of the different constituents that contribute to the PHA granules. The data obtained from these calculations are compared with data from the literature, which show that PHA granules consist not only of the polyester but also of phospholipids and proteins. The latter are referred to as granule-associated proteins, and they are always located at the surface of the PHA granules. A concept is proposed that distinguishes four classes of structurally and functionally different granule-associated proteins: (i) class I comprises the PHA synthases, which catalyze the formation of ester linkages between the constituents; (ii) class II comprises the PHA depolymerases, which are responsible for the intracellular degradation of PHA, (iii) class III comprises a new type of protein, which is referred to as phasins and which has most probably a function analogous to that of oleosins in oilseed plants, and (iv) class IV comprises all other proteins, which have been found to be associated with the granules but do not belong to classes I-III. Particular emphasis is placed on the phasins, which constitute a significant fraction of the total cellular protein. Phasins are assumed to form a close protein layer at the surface of the granules, providing the interface between the hydrophilic cytoplasm and the much more hydrophobic core of the PHA inclusion.

Isolation and purification of granule-associated proteins relevant for poly(3-hydroxybutyric acid) biosynthesis from methylotrophic bacteria relying on the serine pathway

The poly(3-hydroxybutyric acid) (PHB) granules from eight methylotrophic bacteria that use the serine pathway were isolated in a sucrose gradient (1–2 M); these bacteria included members of the genus Methylobacterium, Mycoplana rubra, and PHB-leaky mutants of Methylobacterium rhodesianum. As shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, the granules from all investigated methylotrophic strains revealed two major bands representing small proteins. An efficient purification procedure for these two low molecular weight proteins associated with the PHB granules was developed by solubilization of the proteins with Triton X-114 and affinity chromatography on Procion Blue-H-ERD.Key words: poly(3-hydroxybutyric acid), granule-associated proteins, methylotrophic bacteria.

Granzyme B and Perforin Lytic Proteins Are Expressed in CD34+ Peripheral Blood Progenitor Cells Mobilized by Chemotherapy and Granulocyte Colony-Stimulating Factor

Granzyme B and perforin are cytoplasmic granule-associated proteins used by cytotoxic T lymphocytes and natural killer (NK) cells to kill their targets. However, granzyme B gene expression has also been detected in a non-cytotoxic hematopoietic murine multipotent stem cell line, FDCP-Mix. The objective of the present study was to investigate whether granzyme B and perforin could be expressed in human hematopoietic CD34+ cells and if present, discover what their physiologic relevance could be. The primitive CD34+ human cell line KG1a was investigated first and was found to express granzyme B and perforin. Highly purified hematopoietic stem/progenitor cells were then selected using the CD34 surface antigen as marker. Steady-state bone marrow (BM) CD34+ cells did not contain these proteins. Peripheral blood (PB) CD34+ cells, which had been induced to circulate, were also analyzed. After chemotherapy (CT) and granulocyte colony-stimulating factor (G-CSF) treatment, CD34+ cells strongly expressed mRNAs and proteins of granzyme B and perforin. In contrast, CD34+ cells mobilized by G-CSF alone were negative. Western blot analysis further showed that granzyme B and perforin proteins were identical in CD34+ cells and activated PBLs. Such proteins might be implicated in the highly efficient migration of CD34+ stem/progenitor cells from BM to PB after CT and G-CSF treatment. The cellular adhesion mechanisms involved in the BM homing of CD34+ cells are disrupted at least temporarily after CT. The Asp-ase proteolytic activity of granzyme B on extracellular matrix proteins could be used by progenitor cells for their rapid detachment from BM stromal cells and perforin might facilitate their migration across the endothelial cell barrier.

Serum eosinophil granule-associated protein concentration in primary biliary cirrhosis

Serum eosinophil granule-associated protein concentration in primary biliary cirrhosis

Antibacterial proteins of granulocytes differ in interaction with endotoxin. Comparison of bactericidal/permeability-increasing protein, p15s, and defensins.

Bactericidal/permeability-increasing protein (BPI), antibacterial 15-kDa protein isoforms (p15s), and defensins (neutrophil peptides or NPs) are granule-associated antibacterial proteins of polymorphonuclear leukocytes (PMN) that have both direct and synergistic growth inhibitory activity against Gram-negative bacteria. In this study, we have compared in vitro the abilities of these antibacterial proteins, alone and in combination, to inhibit the endotoxic activity of isolated LPS and whole bacteria. All three proteins blocked endotoxin activity in: 1) the Limulus amoebocyte lysate assay, 2) priming of PMN for enhanced arachidonate release, and 3) stimulating leukocyte oxidase activity in 1% blood. However, the proteins differ markedly in both relative potency (BPI >> p15s = NP1) in the presence of the plasma LPS-binding protein and in the range of LPS chemotypes that can be inhibited. BPI potently neutralizes LPS of any chemotype, but p15s and defensins are less active against long-chain (S-type) LPS. In whole blood ex vivo, the p15s and NP1 are approximately 1000-fold less potent than BPI, but at subinhibitory doses act in synergy with BPI to inhibit the TNF-inducing activity of a serum-resistant encapsulated strain of Escherichia coli (K1/r). The anti-endotoxic effects of p15 and NP1 against E. coli K1/r in whole blood appear secondary to growth arrest, because, in marked contrast to BPI, they are not evident against nonviable bacteria (pretreated with antibiotic) nor isolated LPS. Thus, BPI stands out for its ability to inhibit isolated or bacterial LPS under physiologic conditions. However, p15s and defensins may also contribute to suppression of endotoxic signaling by Gram-negative bacteria via synergistic (with BPI) growth inhibition upon extracellular release of these proteins from PMN during inflammation.

Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules

The function of the polyhydroxyalkanoic acid (PHA) granule-associated GA14 protein of Rhodococcus ruber was investigated in Escherichia coli XL1-Blue, which coexpressed this protein with the polyhydroxybutyric acid (PHB) biosynthesis operon of Alcaligenes eutrophus. The GA14 protein had no influence on the biosynthesis rate of PHB in E. coli XL1-Blue(pSKCO7), but this recombinant E. coli strain formed smaller PHB granules than were formed by an E. coli strain that expressed only the PHB operon. Immunoelectron microscopy with GA14-specific antibodies demonstrated the binding of GA14 protein to these mini granules. In a previous study, two hydrophobic domains close to the C terminus of the GA14 protein were analyzed, and a working hypothesis that suggested an anchoring of the GA14 protein in the phospholipid monolayer surrounding the PHA granule core by these hydrophobic domains was developed (U. Pieper-Fürst, M. H. Madkour, F. Mayer, and A. Steinbüchel, J. Bacteriol. 176:4328-4337, 1994). This hypothesis was confirmed by the construction of C-terminally truncated variants of the GA14 protein lacking the second or both hydrophobic domains and by the demonstration of their inability to bind to PHB granules. Further confirmation of the hypothesis was obtained by the construction of a fusion protein composed of the acetaldehyde dehydrogenase II of A. eutrophus and the C terminus of the GA14 protein containing both hydrophobic domains and by its affinity to native and artificial PHB granules.

Extracellular accumulation of potently microbicidal bactericidal/permeability-increasing protein and p15s in an evolving sterile rabbit peritoneal inflammatory exudate.

To what extent the host defense role of granule-associated antibacterial proteins and peptides of PMN includes extracellular action has not been established. To address this question, we have analyzed the antibacterial activity of cell-free (ascitic) fluid (AF) obtained from glycogen-induced sterile inflammatory rabbit peritoneal exudates in which > 95% of the accumulating cells are PMN. AF, but not plasma collected in parallel, exhibits potent activity toward serum-resistant Gram-negative and Gram-positive bacteria. Total and specific antibacterial activity of AF increases during the first 12 h after injection of glycogen in parallel with the influx of PMN. At maximum, > 99% of 10(7) encapsulated Escherichia coli and Staphylococcus aureus are killed in 30 min/ml of AF. Neutralizing antibodies against the bactericidal/permeability-increasing protein (BPI) of PMN abolishes activity of AF toward encapsulated E. coli but has no effect on activity vs staphylococci. However, BPI alone (approximately 1 microgram/ml in AF) can only account for < or = 20% of AF activity toward E. coli. AF also contains 15 kD PMN proteins (p15s) that act in synergy with BPI. Purified BPI and p15s, in amounts present in AF, reconstitute the growth-inhibitory activity of AF toward encapsulated E. coli. These findings show for the first time an extracellular function of endogenous BPI, providing, together with the p15s, a potent microbicidal system toward Gram-negative bacteria resistant to plasma-derived proteins and phagocytes in inflammatory exudates.

The neuroendocrine precursor 7B2 is a sulfated protein proteolytically processed by a ubiquitous furin-like convertase.

The neuroendocrine granule-associated protein 7B2, unlike many other neuroendocrine precursor proteins stored in secretory granules, carries in its primary structure the Arg-Xaa-Arg/Lys-Arg processing site usually found in constitutively secreted precursor proteins and recognized by the ubiquitously expressed convertase, furin. pro7B2 (30 kDa), when expressed in endocrine (AtT-20, PC12, and GH4C1) or non-endocrine (Ltk-) cell lines using recombinant vaccinia viruses, was converted to a 23-kDa form. Mutation of the P4 Arg to Gly completely prevented this conversion. When excess pro7B2 was coexpressed with the pro-protein convertases PC1, PC2, or furin, only furin could induce complete processing. In addition, coexpression of pro7B2 in LoVo cells, which are devoid of endogenous furin activity, with each one of the three convertases, showed that only furin was able to induce processing of this precursor. pro7B2 processing in AtT-20 was completely abolished when protein transport into Golgi compartments was blocked by cell incubation at either 15 or 37 degrees C in the presence of monensin or brefeldin A. Furthermore, pulse-chase experiments in the presence of Na2[35S]SO4 showed that pro7B2 is Tyr-sulfated in the trans-Golgi network before it is processed. These results demonstrate that pro7B2 is first processed by a furin-like enzyme within the trans-Golgi network into a 23-kDa form that is then sequestered into secretory granules.

Cooperation of Natural Killer Cell Stimulatory Factor/Interleukin-12 with Other Stimuli in the Induction of Cytokines and Cytotoxic Cell-Associated Molecules in Human T and NK Cells

The ability of natural killer cell stimulatory factor/interleukin-12 (IL-12) to induce cytokines other than IFN-γ from both T and NK cells was studied. Both the direct effect of IL-12 and the cooperation between IL-12 and other cytokine inducers such as IL-2, phorbol diesters, and receptor antibodies were evaluated. It was found that IL-12 induces mRNA accumulation and production of GM-CSF and TNF-α from both T and NK cells and, as tested on NK cells only, induced M-CSF mRNA accumulation. Compared with cytokine inducers, IL-12 ability to induce IFN-γ production was severalfold higher than its ability to induce GM-CSF or TNF-γ. Likewise, the synergistic effect of IL-12 with the other stimuli to induce IFN-γ was stronger than that observed in the case of GM-CSF or TNF-α. The IL-12-mediated enhancement of NK cytotoxicity is accompanied by an increased accumulation of mRNA for at least two genes encoding cytotoxic cell granule-associated proteins, the serine esterase granzyme B and the pore-forming protein perforin. IL-12 induction of perforin mRNA accumulation was not synergistic with either IL-2 or anti-CD16 stimulation, whereas granzyme B mRNA accumulation, induced by IL-2 or anti-CD16 stimulation, was slightly potentiated by IL-12. Thus, although IFN-γ production is probably one of the most physiologically relevant effects of IL-12, induction of other cytokines by IL-12 in the presence of other inflammatory or immune stimuli may have a role in the in vivo functions of IL-12. The observation that both the IL-12-mediated enhancement of NK cell-mediated cytotoxicity and the increased expression of genes encoding cytotoxic cell granule-associated proteins were not cooperative with the effect of other NK cell activators suggests that the effect of IL-12 on cytotoxic cells is in part independent from that or other stimuli regulating the functions of these cells.

Intron-exon organization and chromosomal localization of the human TIA-1 gene.

TIA-1 is a 40-kDa cytotoxic granule-associated RNA-binding protein (p40-TIA-1), the expression of which is restricted to cytolytic lymphocytes. The major granule-associated species is a 15-kDa protein (p15-TIA-1) that seems to be derived from the carboxyl terminus of p40-TIA-1. Although some evidence suggests that p15-TIA-1 is derived from p40-TIA-1 by proteolytic processing, it is also possible that each isoform is derived from discrete mRNA initiated from alternative promoters within the TIA-1 gene. To learn more about the relationship between p15-TIA-1 and p40-TIA-1, we have determined the complete intron-exon organization of the TIA-1 gene. The gene consists of 13 exons separated by 12 intervening sequences and spans greater than 46 kb of DNA located on chromosome 2, band p13. The transcription start site of the mRNA transcript encoding p40-TIA-1 was identified by primer extension and S1 mapping analysis. The putative promoter region preceding this transcription start site stimulated the expression of a reporter gene in transfected Jurkat and YT cells. Attempts to identify a second promoter capable of initiating an mRNA encoding p15-TIA-1 were unsuccessful, supporting the possibility that p15-TIA-1 can be derived from p40-TIA-1 proteolytically. Granule-associated serine proteases that have been implicated in cytolytic lymphocyte killing (granzyme A and granzyme B) were unable to cleave TIA-1, suggesting that processing may occur before TIA-1 enters the cytotoxic granule.

Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins.

Vesicular transport within the secretory pathway can be arrested by incubating cells at 15 degrees C or 20 degrees C to block exit from the endoplasmic reticulum or trans-Golgi network, respectively. Using this powerful tool we have compared the intracellular sites of endoproteolytic processing of proopiomelanocortin and two prohormone processing enzymes in AtT-20 mouse pituitary corticotrope tumor cells. For comparison, proopiomelanocortin processing was also evaluated in primary neurointermediate pituitary cultures. AtT-20 cells synthesize and store endogenous proopiomelanocortin and prohormone convertase 1; AtT-20 cells expressing high levels of integral membrane or soluble peptidylglycine alpha-amidating monooxygenase were generated by stable transfection. Cells were incubated with [35S]methionine and chased at 4 degrees C, 15 degrees C, 20 degrees C or 37 degrees C. The endoproteolytic processing of peptidylglycine alpha-amidating mono-oxygenase, prohormone convertase 1, and proopiomelanocortin was compared following immunoprecipitation. Endoproteolytic processing of integral membrane and soluble peptidylglycine alpha-amidating monooxygenase proteins was completely blocked by incubation of cells at 20 degrees C. In contrast, prohormone convertase 1 processing from the 87 kDa precursor to the 81 kDa intermediate proceeded to completion at both 15 degrees C and 20 degrees C, while cleavage to generate the 63 kDa prohormone convertase 1 protein was completely blocked at 20 degrees C. In AtT-20 cells and neurointermediate pituitary cultures, generation of beta-lipotropin from proopiomelanocortin continued at a slow but significant rate at 20 degrees C, while processing of beta-lipotropin to beta-endorphin was blocked. Thus prohormone convertase 1 processing begins in the endoplasmic reticulum and is not completed until after the trans-Golgi network, while peptidylglycine alpha-amidating monooxygenase processing begins after the trans-Golgi network. Selected proopiomelanocortin cleavages begin before entry into immature granules.

Differential trafficking of soluble and integral membrane secretory granule-associated proteins

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.