High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.     

Evidence for a granule targeting sequence within platelet factor 4

Platelets achieve bleeding arrest at sites of vascular injury via secretion of secretory proteins from their storage granules, termed alpha-granules. We have recently analyzed granule targeting of platelet factor 4 (PF4), a secretory alpha-granule chemokine, and demonstrated that PF4 alpha-granule storage relied upon determinants within PF4 mature sequence. To define these determinants, PF4 mutants fused to the fluorescent reporter protein green fluorescent protein were generated by progressive deletions and site-directed mutagenesis. They were then transfected in AtT20 cells and assessed for granule targeting by colocalization with ACTH-containing granules, using laser scanning confocal microscopy. This strategy identified the amino acid 41-50 (LIATLKNGRK) sequence as most critical for PF4 granule targeting and/or storage; its deletion from PF4 induced a marked decrease in granule storage (from 81 +/- 2% to 17 +/- 3%, p < or = 0.0001). Ala-scanning mutagenesis of LIATLKNGRK narrowed down the targeting motif to LKNG. A direct role for LKNG in alpha-granule targeting was confirmed in the thrombopoietin-induced human megakaryocytic Dami cells, in which the LKNG-green fluorescent protein chimera exhibited an 82.5 +/- 1.8% colocalization with the alpha-granule proteins von Willebrand factor and P-selectin. LKNG is poorly conserved within the chemokine family. However three-dimensional alignments of the human alpha-granule chemokines Nap-2 (neutrophil-activating peptide) and RANTES (Regulated upon Activation Normal T Cell Expressed and Secreted) with PF4 revealed that LKNG, a surface-exposed hydrophilic turn/loop, matched Nap-2 (LKDG) and RANTES (TRKN) peptides with similar features. Moreover Nap-2 and RANTES peptides exhibited the same alpha-granule targeting efficiency than LKNG. We therefore postulate that the three-dimensional and physicochemical characteristics of PF4 LKNG are of general relevance to alpha-granule targeting of chemokines and possibly of other alpha-granule proteins.     

Sorting ourselves out: seeking consensus on trafficking in the beta-cell

Biogenesis of the regulated secretory pathway in the pancreatic beta-cell involves packaging of products, notably proinsulin, into immature secretory granules derived from the trans -Golgi network. Proinsulin is converted to insulin and C-peptide as granules mature. Secretory proteins not entering granules are conveyed by transport intermediates directly to the plasma membrane for constitutive secretion. One of the co-authors, Peter Arvan, has proposed that in addition, small vesicles bud from granules to traffic to the endosomal system. From there, some proteins are secreted by a (post-granular) constitutive-like pathway. He argues that retention in granules is facilitated by condensation, rendering soluble products (notably C-peptide and proinsulin) more available for constitutive-like secretion. Thus he argues that prohormone conversion is potentially important in secretory granule biogenesis. The other co-author, Philippe Halban, argues that the post-granular secretory pathway is not of physiological relevance in primary beta-cells, and contests the importance of proinsulin conversion for retention in granules. Both, however, agree that trafficking from granules to endosomes is important, purging granules of unwanted newly synthesized proteins and allowing their traffic to other destinations. In this Traffic Interchange, the two co-authors attempt to reconcile their differences, leading to a common vision of proinsulin trafficking in primary and transformed cells.     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.     

Regulation of peptidergic vesicle mobility by secretagogues

Neuropeptides are released into the extracellular space from large secretory granules. In order to reach their release sites, these granules are translocated on microtubules and thought to interact with filamentous actin as they approach the cell membrane. We have used a green fluorescent protein-tagged neuropeptide prohormone (prepro-orphanin FQ) to visualize vesicle trafficking dynamics in NS20Y cells and cultures of primary hippocampal neurons. We found that the majority of secretory granules were mobile and accumulated at both the tips of neurites as well as other apparently specialized cellular sites. We also used live-cell imaging to test the notion that peptidergic vesicle mobility was regulated by secretagogues. We show that treatment with forskolin appeared to increase vesicle rates of speed, while depolarization with high K⁺ had no effect, even though both treatments stimulated neuropeptide secretion. In cultured hippocampal neurons the green fluorescent protein-tagged secretory vesicles were routed to both dendrites and axons, indicating that peptidergic vesicle transport was not polarized. Basal peptidergic vesicle mobility rates in hippocampal neurons were the same as those in NS20Y cells. Taken together, these studies suggest that secretory vesicle mobility is regulated by specific classes of secretagogues and that neuropeptide containing secretory vesicles may be released from dendritic structures.     

VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion.

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta-cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP-2 and cellubrevin, but not VAMP-1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta-cells contain secretory granules that store and secrete insulin as well as synaptic-like microvesicles carrying gamma-aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP-2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP-2 with insulin-containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta-cells. Pretreatment of streptolysin-O permeabilized insulin-secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP-2 and cellubrevin and abolished Ca(2+)-induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S-stimulated insulin secretion. Taken together, our results show that pancreatic beta-cells express VAMP-2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)-mediated insulin secretion.     

Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.     

Real-Time Imaging of the Axonal Transport of Granules Containing a Tissue Plasminogen Activator/Green Fluorescent Protein Hybrid

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.     

Protein discharge from immature secretory granules displays both regulated and constitutive characteristics.

At physiological glucose concentrations, isolated pancreatic islets release a minor portion of their newly synthesized insulin and precursors in a phase of secretion which is largely complete by 4 h of chase. Discharge during this period can be amplified by secretagogues, yet is not abolished by conditions which fully suppress regulated release from dense core secretory granules. The ability to stimulate the secretion and the biochemical profile of released proinsulin-related peptides indicate that secretion during this period originates from immature granules. The stoichiometry of release of labeled C-peptide:insulin during this phase is 1:1 at high glucose concentrations. However, at physiologic or low concentrations, C-peptide is released in molar excess of insulin as if the exocytotic vesicles carrying this secretion were budding from a post-Golgi compartment in which the lumen was composed of condensing insulin and soluble C-peptide. These findings can be explained by a model for regulated secretory protein traffic in which direct exocytosis of young granules is stimulated by higher glucose concentrations and vesicle budding from immature granules occurs at lower concentrations. Thus, insulin targeting from immature granules exhibits both regulated and constitutive-like characteristics.     

Preferential Release of Newly Synthesized Insulin Assessed by a Multi-Label Reporter System Using Pancreatic β-Cell Line MIN6

Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic β-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.     

EGFP-Tagged Vasopressin Precursor Protein Sorting Into Large Dense Core Vesicles and Secretion From PC12 Cells

1. Hypothalamic magnocellular neurons synthesize, store, and secrete large quantities of the neuropeptides, vasopressin (VP) and oxytocin (OT), which are synthesized as protein precursors also containing proteins called neurophysins. These protein precursors are sorted through the regulated secretory pathway (RSP), packaged into large dense core vesicles LDCVs, and their peptide products are secreted from nerve terminals in the posterior pituitary.2. It has been hypothesized that this efficient packaging is dependent on the interaction of the peptide with neurophysin in a complex that forms the granule core. To test this, PC12 cells were transfected with vasopressin precursor DNA constructs that either contained or deleted the neurophysin moiety and tagged with enhanced green fluorescent protein (EGFP) as reporters. The intracellular routing and secretion of the EGFP-tagged VP precursor proteins were studied by in differentiated PC12 cells by fluorescence microscopy, electron microscopic immunocytochemistry, and fluorescent imaging techniques.3. The data showed that only when the neurophysin was present in the VP precursor construct did the fluorescent fusion protein become routed to the RSP and get efficiently packaged into LDCVs and secreted. These data are consistent with the view that routing of the precursor to LDCVs requires the amino acids that encode the intravesicular chaperone, neurophysin.     

Mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells

Enteropeptidase, a type II transmembrane protein of the enterocyte brush border, is sorted directly to the apical membrane of Madin-Darby canine kidney II cells. Apical targeting appears to be mediated by an N-terminal segment that contains a 27-amino acid residue O-glycosylated mucin-like domain consisting of two short mucin-like repeats, A and B. Targeting signals within these repeats were characterized by using green fluorescent protein (GFP) as a reporter. Constructs with a cleavable signal peptide and both repeats A and B were secreted apically. Similar constructs lacking mucin repeats were secreted randomly. Either repeat A or B was sufficient to direct apical targeting of GFP. O-linked oligosaccharides alone were not sufficient for targeting because fusion to a different O-glycosylated motif did not alter the random secretion of GFP, and several constructs with mutations in either repeat A or B were O-glycosylated and secreted randomly. In addition, repeat B appears to contain an apical targeting signal that functions in the absence of glycosylation. Density gradient centrifugation indicated that, unlike several other apically targeted membrane and soluble proteins, apical sorting of mucin-GFP chimeric proteins does not appear to utilize lipid rafts.     

5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics

Changes in 5'-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761-774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 beta-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKalpha1, 1-312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mM) vesicle movements, and decreased the number of vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent vesicle movements. These findings suggest that components of the vesicle transport network, including vesicle-associated motor proteins, may be targets of AMPK in beta-cells, dephosphorylation of which is required for vesicle mobilization at elevated glucose concentrations.     

New roles of myosin II during vesicle transport and fusion in chromaffin cells

Modified herpes virus (amplicons) were used to express myosin regulatory light chain (RLC) chimeras with green fluorescent protein (GFP) in cultured bovine chromaffin cells to study myosin II implication in secretion. After infection, RLC-GFP constructs were clearly identified in the cytoplasm and accumulated in the cortical region, forming a complex network that co-localized with cortical F-actin. Cells expressing wild type RLC-GFP maintained normal vesicle mobility, whereas cells expressing an unphosphorylatable form (T18A/S19A RLC-GFP) presented severe restrictions in granule movement as measured by individual tracking in dynamic confocal microscopy studies. Interestingly, the overexpression of this mutant form of RLC also affected the initial secretory burst elicited by either high K(+) or BaCl(2), as well as the secretion induced by fast release of calcium from caged compounds in individual cells. Moreover, T18A/S19A RLC-GFP-infected cells presented slower fusion kinetics of individual granules compared with controls as measured by analysis of amperometric spikes. Taken together, our results demonstrate the implication of myosin II in the transport of vesicles, and, surprisingly, in the final phases of exocytosis involving transitions affecting the activity of docked granules, and therefore uncovering a new role for this cytoskeletal element.     

Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.     

The green fluorescent protein targets secretory proteins to the yeast vacuole

The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNDeltaVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNDeltaVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.     

Quinacrine accumulates in certain peptide hormone-producing cells

Summary Quinacrine is a fluorescent anti-malarial acridine derivative which binds selectively to a population of nerves, presumably peptidergic, and to certain peptide hormone-producing cells. Among these cells are glycopeptide hormone-producing cells in the adenohypophysis, the calcitonin cells in the thyroid, the insulin, glucagon and PP cells in the pancreatic islets, and the gastrin cells in the pyloric antrum. Available evidence suggests that the fluorophore accumulates in the secretory granules. The half-life of the fluorescence varies from one cell type to another, from 6 h in the gastrin cells to 40 h in the insulin cells. It cannot be excluded that the half-life of the fluorescence reflects the turn-over rate of the secretory granules and that the disappearance rate of the fluorescence is dependent upon the secretory activity of the cell.