P-selectin Targeting to Secretory Lysosomes of Rbl-2H3 Cells
2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês
10.1074/jbc.m111293200
ISSN1083-351X
AutoresJasber Kaur, Daniel F. Cutler,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes. The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes. synaptic like microvesicles Weibel-Palade bodies Dulbecco's modified Eagle's medium horseradish peroxidase phosphate-buffered saline post-nuclear supernatant wild type tyramide signal amplification Secretory lysosomes are a distinct class of regulated secretory organelle. Not only does this organelle serve as the final degradative compartment of the cell, but it also stores secretory molecules that are released in response to an extracellular trigger. This exocytic capacity clearly marks them from conventional lysosomes. Although conventional lysosomes can also fuse with the plasma membrane and release their soluble contents following stimulation (1Andrews N. Trends Cell Biol. 2000; 10: 316-321Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar), the extent of Ca2+-triggered secretion of lysosomal enzymes from cells such as fibroblasts and epithelial cells tends to be only 10–20% (2Rodriguez A. Webster P. Ortego J. Andrews N. J. Cell Biol. 1997; 137: 93-104Crossref PubMed Scopus (414) Google Scholar). In comparison, up to 80% of lysosomal markers are released upon a physiological trigger from cells that possess secretory lysosomes. Cells such as cytotoxic T lymphocytes, neutrophils, melanocytes, mast cells, and basophils use their secretory lysosomes to store specialized components such as granzymes, melanin, histamine, and serotonin, in addition to their usual lysosomal content (3Dell'Angelica E.C. Mullins C. Caplan S. Bonifacino J.S. FASEB J. 2000; 14: 1265-1278Crossref PubMed Google Scholar, 4Stinchcombe J.C. Griffiths G.M. J. Cell Biol. 1999; 147: 1-5Crossref PubMed Scopus (127) Google Scholar, 5Griffiths G.M. Trends Cell Biol. 1996; 6: 329-332Abstract Full Text PDF PubMed Scopus (96) Google Scholar). Rbl-2H3 is a basophilic leukemia cell line that has been extensively used in studies of regulated exocytosis. Morphological and biochemical studies have revealed that Rbl-2H3 secretory granules possess an acidic pH, contain mature lysosomal enzymes, and are accessible to endocytic markers, all hallmarks of lysosomes, but they also contain secretory markers such as serotonin. Both "lysosomal" and "granule" markers can be released upon stimulation of the cells by aggregation of FcεRI (6Dragonetti A. Baldassarre M. Castino R. Demoz M. Luini A. Buccione R. Isidoro C. J. Cell Sci. 2000; 113: 3289-3298Crossref PubMed Google Scholar, 7Xu K. Williams R.M. Holowka D. Baird B. J. Cell Sci. 1998; 111: 2385-2396Crossref PubMed Google Scholar). Thus the endocytic and exocytic apparatus are linked such that the lysosome has been modified to become the regulated secretory organelle of these cells. Given the hybrid nature of the secretory lysosome, an interesting question is which cytoplasmic targeting signals (in this paper we define a targeting signal as a peptide sequence that is required for the accumulation of a protein within an organelle) direct membrane proteins to this organelle. The simplest possibility is that lysosomal targeting sequences operate to direct both lysosomal and secretory membrane proteins to the modified lysosome (3Dell'Angelica E.C. Mullins C. Caplan S. Bonifacino J.S. FASEB J. 2000; 14: 1265-1278Crossref PubMed Google Scholar). Alternatively, secretory granule or entirely novel targeting signals that are specific to secretory lysosomes might be used. When tyrosinase, the resident membrane protein of melanosomes is heterologously expressed in HeLa and Madin-Darby canine kidney cells, it localizes to lysosomes in a di-leucine signal-dependent manner (8Calvo P.A. Frank D.W. Bieler B.M. Berson J.F. Marks M.S. J. Biol. Chem. 1999; 274: 12780-12789Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 9Simmen T. Schmidt A. Hunziker W. Beermann F. J. Cell Sci. 1999; 112: 45-53PubMed Google Scholar). In addition this di-leucine signal mediates targeting of tyrosinase to synaptic like microvesicles (SLMV)1 from the endocytic pathway of PC12 cells (10Blagoveshchenskaya A.D. Hewitt E.W. Cutler D.F. Mol. Biol. Cell. 1999; 10: 3979-3990Crossref PubMed Scopus (60) Google Scholar). Thus tyrosinase appears to possess the necessary sorting information for its localization to secretory lysosomes, conventional lysosomes, and SLMV. In contrast, it has been suggested that Fas ligand is sorted to the secretory lysosome of cytotoxic T lymphocytes using novel signals in its cytoplasmic tail, specific to this class of organelle, because when heterologously expressed, Fas ligand localizes only to secretory lysosomes (Rbl-2H3 cells) but not to conventional lysosomes (HeLa cells) (11Bossi G. Griffiths G.M. Nat. Med. 1999; 5: 90-96Crossref PubMed Scopus (328) Google Scholar). P-selectin resides in the membranes of α and δ granules of platelets (12Berman C.L. Yeo E.L. Wencel-Drake J.D. Furie B.C. Ginsberg M.H. Furie B. J. Clin. Invest. 1986; 78: 130-137Crossref PubMed Scopus (357) Google Scholar, 13Stenberg P.E. McEver R.P. Shuman M.A. Jacques Y.V. Bainton D.F. J. Cell Biol. 1985; 101: 880-886Crossref PubMed Scopus (746) Google Scholar, 14Israels S.J. Gerrard Y.V. Jacques Y.V. McNicol A. Cham B. Nishibori M. Bainton D.F. Blood. 1992; 80: 143-152Crossref PubMed Google Scholar) and also in Weibel-Palade bodies (WPB) (15Bonfanti R. Furie B.C. Furie B. Wagner D.D. Blood. 1989; 73: 1109-1112Crossref PubMed Google Scholar, 16McEver R.P. Beckstead J.H. Moore K.L. Marshall-Carlson L. Bainton D.F. J. Clin. Invest. 1989; 84: 92-99Crossref PubMed Scopus (844) Google Scholar) and lysosomes (17Arribas M. Cutler D.F. Traffic. 2000; 1: 783-793Crossref PubMed Scopus (38) Google Scholar) of endothelial cells. The short, 35-amino acid cytoplasmic tail of P-selectin is necessary for targeting to WPB, but surprisingly, deletion of this region does not affect sorting to the secretory lysosomal α granules of platelets (18Hartwell D.W. Mayadas T.N. Berger G. Frenette P.S. Rayburn H. Hynes R.O. Wagner D.D. J. Cell Biol. 1998; 143: 1129-1141Crossref PubMed Scopus (97) Google Scholar). Importantly, heterologous expression of P-selectin has allowed for the definition of both granule and lysosomal targeting signals, as well as those for delivery to SLMV, in a variety of cell types (Fig.1). It is therefore an ideal candidate to investigate the following two questions: first, is the cytoplasmic tail of P-selectin sufficient for delivery to secretory lysosomes of Rbl-2H3 cells, and second, if so, then does delivery rely on granule, lysosomal, or novel targeting signals? We have combined subcellular fractionation coupled to transient transfection of HRP-P-selectin chimeras in Rbl-2H3 cells to answer these questions. Micro-BCA Protein Assay Reagent kit was used according to the manufacturer's instructions (Pierce). [3H]Serotonin (hydroxytryptamine binoxylate-5), specific activity 28 Ci/mm, was purchased from PerkinElmer Life Sciences.125I-Diferric transferrin (human), specific activity 60 Ci/mm, was purchased from PerkinElmer Life Sciences. TSA Fluorescence Systems (cyanine 3) was purchased from PerkinElmer Life Sciences. All chemicals were purchased from Sigma, unless otherwise stated. HRP-P-selectin is a chimera comprising the human growth hormone signal sequence, followed by horseradish peroxidase (HRP) and the transmembrane and cytoplasmic domains of P-selectin in pRK34 (18Hartwell D.W. Mayadas T.N. Berger G. Frenette P.S. Rayburn H. Hynes R.O. Wagner D.D. J. Cell Biol. 1998; 143: 1129-1141Crossref PubMed Scopus (97) Google Scholar). 763HRP-P-selectin is a truncation of the cytoplasmic tail (21Norcott J.P. Solari R. Cutler D.F. J. Cell Biol. 1996; 134: 1229-1240Crossref PubMed Scopus (52) Google Scholar), and KCPL, YGVF,DPSPHRP-P-selectin are tetra-alanine mutations as described (24Blagoveshchenskaya A.D. Norcott J.P. Cutler D.F. J. Biol. Chem. 1998; 273: 2729-2737Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Blagoveshchenskaya A.D. Hewitt E.W. Cutler D.F. J. Biol. Chem. 1998; 273: 27896-27903Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) (see Fig. 3). Rbl-2H3 cells (kind gift from Mark Marsh, MRC Unit, LMCB, University College London) were grown as an adherent monolayer in DMEM (Life Technologies, Inc.), supplemented with 10% heat-inactivated fetal bovine serum, 50 μg/ml Gentamicin (Life Technologies, Inc.), at 37 °C, 5% CO2. For transient transfection studies, ∼4 × 107 cells were electroporated (three pulses) at 125 microfarads, 250 V, infinity ohms. For wt, YGVF,DPSPHRP-P-selectin constructs, 10 μg of DNA was used for transfection, whereas 3 μg of DNA was used for KCPL,763HRP-P-selectin constructs to obtain similar levels of expression. Cells were replated in their usual growth medium and analyzed 3 days post-transfection. Cells were primed overnight (day 2 post-transfection) with 0.5 μg/ml anti-2,4-dinitrophenol IgE (monoclonal, SPE-7, Sigma) and also incubated with 0.2 μCi/ml [3H]serotonin (hydroxytryptamine binoxylate-5, PerkinElmer Life Sciences) in growth medium prior to stimulation the following day. Cells were rinsed twice and cultured for 1 h in normal media. Stimulation was induced by the addition of 50 ng/ml human serum albumin/2,4-dinitrophenol to cross-link the IgE bound to FcεRI on the cell surface for 25 min at 37 °C. This took place in DMEM (without serum, 10 mm HEPES, 2 mg/ml bovine serum albumin) with no phenol red (to prevent interference of absorbance assays for β-hexosaminidase). Cells were then placed on ice, and the stimulation medium was removed and kept for analysis. Three days post-transfection, cells were serum-starved (DMEM, 10 mmHEPES, 2 mg/ml bovine serum albumin) for 1 h and incubated with 0.6 μCi/ml 125I-transferrin for 1 h. Cells were then rinsed three times in cold DMEM. To locate the plasma membrane, cell surface 125I-transferrin was stripped by the following method. Following loading with 125I-transferrin, cells were transferred to ice, rinsed, and incubated for 15 min in 20 mm sodium acetate, pH 5, 2 mmCaCl2, 150 mm NaCl, and 50 μmdeferoxamine mesylate. This was replaced with PBS+ (PBS, 1 mm MgCl2, 0.1 mm CaCl2) and then further rinsed and incubated for 20 min with PBS+ and 50 μm deferoxamine mesylate. Single sucrose gradient subcellular fractionation was performed according to a procedure modified from that published by Baram et al. (27Baram D. Adachi R. Medalia O. Tuvim M. Dickey B.F. Mekori Y.A. Sagi-Eisenberg R. J. Exp. Med. 1999; 189: 1649-1657Crossref PubMed Scopus (102) Google Scholar). Cells were placed on ice and rinsed twice with homogenization buffer (HB, pH 7.3, 0.25 m sucrose, 1 mm MgCl2, 10 mm HEPES). Samples were scraped (4 × 107) in 1.5-ml volume of HB (plus 1 mm phenylmethylsulfonyl fluoride and protease inhibitor mixture). The cell suspension was homogenized by five passages through a ball bearing homogenizer with 0.009 mm clearance (EMBL, Heidelberg, Germany). The nuclear fraction was spun down at 170 × g for 10 min at 4 °C and 800 units/ml DNase (type IV) added to the post-nuclear supernatant (PNS). The PNS was loaded onto a preformed continuous sucrose gradient as described (27Baram D. Adachi R. Medalia O. Tuvim M. Dickey B.F. Mekori Y.A. Sagi-Eisenberg R. J. Exp. Med. 1999; 189: 1649-1657Crossref PubMed Scopus (102) Google Scholar). Gradients were collected in 25 fractions of 500 μl from the top of the tube using an Autodensi-Flow IIC (Buchler Instruments, Kansas City, MO) and analyzed when necessary for [3H]serotonin, 125I-transferrin, β-hexosaminidase, and HRP activities across the gradient. Recovery of β-hexosaminidase and HRP activities on the primary gradient was ∼50% of total activity (homogenate + medium) in resting cells; however, for stimulated cells, recovery was reduced to around 40% of total activity. For experiments that required a further secondary gradient, fractions 14–20 were analyzed and the remainder pooled to 4 ml at 1.1 m sucrose. The pooled fractions were layered onto a preformed continuous sucrose gradient of 1.3–2.0 msucrose (8 ml). This gradient was then run, fractionated, and analyzed as with the first gradient. [3H]Serotonin incorporation was monitored by tritium radioactivity using liquid scintillation spectrometry. 125I-Transferrin loading was monitored by gamma counting using a Packard Cobra II auto-gamma counter. β-Hexosaminidase activity was determined using an absorbance assay in a microplate reader as follows. 50 μl of sample was mixed with 20 μl of subcellular fractionation buffer (1 mmNaHCO3, 1 mm EDTA, 0.01% Triton X-100). This was then incubated with 100 μl of substrate solution consisting of 4 mg/mlp-nitrophenyl-N-acetyl-β-d-glucosaminide in 0.1 m sodium citrate buffer, (pH 4.5, 0.2% Triton X-100) for 30 min at 37 °C in the dark. The reaction was stopped by the addition of 150 μl of pre-warmed (37 °C) stop buffer (0.25m glycine, 0.2 m NaCl, 4% SDS, pH 12.5). The samples were read at 405 nm in a Molecular Devices Thermo.max microplate reader. HRP activities were determined from aliquots of 75 μl as described previously (28Blagoveshchenskaya A.D. Cutler D.F. Methods Enzymol. 2000; 327: 45-60Crossref PubMed Scopus (5) Google Scholar). Transfected cells on glass coverslips 1 day post-transfection were paraformaldehyde (3%)-fixed, quenched with 50 mm NH4Cl, and rinsed with PBS. Coverslips were then placed in cyanine 3-tyramide signal amplification (TSA) reagent for 10 min. The reaction was stopped with 8 washes in PBS plus azide (0.2%). Samples were permeabilized in 0.2% saponin and then transferred to PBS plus 0.02% saponin and 0.2% gelatin for subsequent antibody incubations. Cells were incubated with mouse monoclonal anti-serotonin (Biogenesis, Poole, UK), followed by goat anti-mouse fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories, West Grove, PA). After staining, cells were mounted in Mowiol and analyzed with the use of an Optiphot-2 microscope (Nikon, Tokyo, Japan) equipped with an MRC Bio-Rad 1024 confocal laser scanning system. Images were transferred to Adobe Photoshop (Adobe Systems, Mountain View, CA). The partitioning of membrane-associated HRP and soluble (clipped) HRP (28Blagoveshchenskaya A.D. Cutler D.F. Methods Enzymol. 2000; 327: 45-60Crossref PubMed Scopus (5) Google Scholar) was established using the Triton X-114 assay (29Masterton W.J. Magee A.I. Magee A.I. Wileman T. Protein Targeting, A Practical Approach. Oxford University Press, New York1992: 242Google Scholar) with the addition of a protease inhibitor mixture upon cell lysis. Transfected and mock-transfected cells were separated into upper and lower phases, and HRP assays were carried out in triplicate. To normalize for differential cell number between samples, the protein concentration (Micro BCA Protein Assay Reagent kit, Pierce) of each phase was determined. For transfected and mock-transfected cells, the HRP activity in each phase (upper or lower) was calculated per μg of total protein (upper plus lower). Levels of HRP activity/μg of protein in the two phases of mock-transfected cells was used to subtract background levels of endogenous peroxidase activity away from transfected cells. The extent of proteolysis of HRP from its P-selectin membrane-bound anchor was established by calculating the percentage of soluble (clipped) HRP in the upper phase to the total of both phases. To determine which targeting signals are required by HRP-P-selectin for accumulation within Rbl-2H3 secretory lysosomes, we combined transient expression with subcellular fractionation, a strategy that has proved highly successful in previous analyses of targeting (21Norcott J.P. Solari R. Cutler D.F. J. Cell Biol. 1996; 134: 1229-1240Crossref PubMed Scopus (52) Google Scholar, 22Blagoveshchenskaya A.D. Hewitt E.W. Cutler D.F. J. Cell Biol. 1999; 145: 1419-1433Crossref PubMed Scopus (40) Google Scholar, 23Blagoveshchenskaya A.D. Cutler D.F. Mol. Biol. Cell. 2000; 11: 1801-1814Crossref PubMed Scopus (21) Google Scholar, 24Blagoveshchenskaya A.D. Norcott J.P. Cutler D.F. J. Biol. Chem. 1998; 273: 2729-2737Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Blagoveshchenskaya A.D. Hewitt E.W. Cutler D.F. J. Biol. Chem. 1998; 273: 27896-27903Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 28Blagoveshchenskaya A.D. Cutler D.F. Methods Enzymol. 2000; 327: 45-60Crossref PubMed Scopus (5) Google Scholar). Because morphological co-localization of Rbl-2H3 endogenous markers such as the granule membrane protein 5G10 (30Bonifacino J.S. Yuan L. Sandoval I.V. J. Cell Sci. 1989; 92: 701-712PubMed Google Scholar), serotonin, lgp120, and β-hexosaminidase has been reported within a single population of organelles (6Dragonetti A. Baldassarre M. Castino R. Demoz M. Luini A. Buccione R. Isidoro C. J. Cell Sci. 2000; 113: 3289-3298Crossref PubMed Google Scholar, 7Xu K. Williams R.M. Holowka D. Baird B. J. Cell Sci. 1998; 111: 2385-2396Crossref PubMed Google Scholar, 31Dell'Angelica E.C. Payne G.S. Cell. 2001; 106: 395-398Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), we expected to find a single peak containing these markers by fractionation. We have chosen two markers for the secretory lysosome, the lysosomal enzyme β-hexosaminidase and the secretory monoamine serotonin (5-hydroxytryptamine), to reflect the twin characteristics of the organelle. We assayed for endogenous activity of β-hexosaminidase and labeled cells with [3H]serotonin. We first established that the uptake of [3H]serotonin was specific by using reserpine, which blocks the vesicular uptake of catecholamines and serotonin (data not shown). Fig.2 A shows the subcellular distribution of the secretory lysosome marker β-hexosaminidase across a single sucrose gradient following centrifugation of the PNS from resting and cells stimulated by cross-linking the IgE receptor (FcεRI) for 25 min. The lysosomal enzyme β-hexosaminidase distributes as a single peak of activity, highlighted in gray (fractions 14–20), and activity falls from this peak by 45% upon stimulation. This fractionation scheme was adapted from the work of Baram et al. (27Baram D. Adachi R. Medalia O. Tuvim M. Dickey B.F. Mekori Y.A. Sagi-Eisenberg R. J. Exp. Med. 1999; 189: 1649-1657Crossref PubMed Scopus (102) Google Scholar), who enriched for secretory lysosomes from Rbl cells using this single sucrose gradient. We have observed the secretory marker [3H]serotonin to always co-distribute with β-hexosaminidase across the gradient in resting cells and to respond to stimulation in the same way as β-hexosaminidase in all experiments carried out to date. [3H]Serotonin, however, was not monitored at this early stage to retain material for subsequent analysis. These same resting and stimulated cells had been transiently transfected 3 days prior with wt HRP-P-selectin (21Norcott J.P. Solari R. Cutler D.F. J. Cell Biol. 1996; 134: 1229-1240Crossref PubMed Scopus (52) Google Scholar) to determine whether the cytoplasmic domain of P-selectin has the necessary sorting information required to direct it to the secretory lysosome of these cells. Fig. 2 Bshows that wt HRP-P-selectin peaks in the same fractions (15Bonfanti R. Furie B.C. Furie B. Wagner D.D. Blood. 1989; 73: 1109-1112Crossref PubMed Google Scholar, 16McEver R.P. Beckstead J.H. Moore K.L. Marshall-Carlson L. Bainton D.F. J. Clin. Invest. 1989; 84: 92-99Crossref PubMed Scopus (844) Google Scholar, 17Arribas M. Cutler D.F. Traffic. 2000; 1: 783-793Crossref PubMed Scopus (38) Google Scholar, 18Hartwell D.W. Mayadas T.N. Berger G. Frenette P.S. Rayburn H. Hynes R.O. Wagner D.D. J. Cell Biol. 1998; 143: 1129-1141Crossref PubMed Scopus (97) Google Scholar, 19Disdier M. Morrisey J.H. Fugate R.D. Bainton D.F. McEver R.P. Mol. Biol. Cell. 1992; 3: 309-321Crossref PubMed Scopus (128) Google Scholar) that contain the marker β-hexosaminidase in resting cells and that this compartment also responds to stimulation of the cells caused by aggregation of the FcεRI for 25 min. Thus HRP-P-selectin is targeted to the functionally active secretory lysosome. A more detailed stimulation profile was carried out by inducing exocytosis for 5 and 10 min to ascertain whether a significant proportion of HRP-P-selectin would shift out of the secretory lysosome peak into other organellar fractions, such as the plasma membrane, upon degranulation of the cells. HRP activity does appear as a shoulder to the main secretory lysosome peak from fractions 9–14, within which the HRP activity increases sequentially as we increase the stimulation period. However, the secretory lysosome peak remains the main peak of HRP activity. Following 5 min of stimulation, 29% HRP activity is lost from the secretory lysosome fractions (14Israels S.J. Gerrard Y.V. Jacques Y.V. McNicol A. Cham B. Nishibori M. Bainton D.F. Blood. 1992; 80: 143-152Crossref PubMed Google Scholar, 15Bonfanti R. Furie B.C. Furie B. Wagner D.D. Blood. 1989; 73: 1109-1112Crossref PubMed Google Scholar, 16McEver R.P. Beckstead J.H. Moore K.L. Marshall-Carlson L. Bainton D.F. J. Clin. Invest. 1989; 84: 92-99Crossref PubMed Scopus (844) Google Scholar, 17Arribas M. Cutler D.F. Traffic. 2000; 1: 783-793Crossref PubMed Scopus (38) Google Scholar, 18Hartwell D.W. Mayadas T.N. Berger G. Frenette P.S. Rayburn H. Hynes R.O. Wagner D.D. J. Cell Biol. 1998; 143: 1129-1141Crossref PubMed Scopus (97) Google Scholar, 19Disdier M. Morrisey J.H. Fugate R.D. Bainton D.F. McEver R.P. Mol. Biol. Cell. 1992; 3: 309-321Crossref PubMed Scopus (128) Google Scholar, 20Modderman P.W. Beuling E.A. Govers L.A.T. Calafat J. Janssen H. Von Dem Borne A.E. Sonnenberg A. Biochem. J. 1998; 336: 153-161Crossref PubMed Scopus (26) Google Scholar); 10 min of stimulation results in a 35% fall, and after 25 min of stimulation there is a 38% fall in HRP activity from the secretory lysosome. This sequential loss of activity from the peak of the secretory lysosome upon stimulation coincides with the sequential increase in activity in fractions 9–14. Following the full 25-min stimulation period, we only observe a 38% fall in HRP activity within the secretory lysosome, whereas the extent of release of β-hexosaminidase following the same period was 45%. To investigate the residual HRP activity within the secretory lysosome peak, we pooled fractions 14–20 (highlighted) and layered them onto a more shallow secondary sucrose gradient (1.3–2.0 m). Fig.2 C shows the distribution of β-hexosaminidase and [3H]serotonin from the pooled fractions of Fig.2 A. The profile of the two markers in resting cells reveals two peaks, the lighter one distributing between fractions 9 and 10 and the denser one between fractions 17 and 20, both of which are responsive when the cells are induced to exocytose for 25 min. Hence both peaks are secretory lysosomes. The traces for resting and stimulated [3H]serotonin show a significant number of counts in the load of the gradient (fractions 1–5) which probably reflect leakage of this tracer molecule from vesicular structures. This is also observed when [3H]serotonin is monitored on the primary gradient (data not shown). Fig. 2 D shows that the resting peaks of HRP-P-selectin activity co-distribute with those for the secretory lysosome markers (2C) and that they also both respond to stimulation for 25 min, HRP activity falling from the secretory lysosome peaks and redistributing to a new lighter peak at fraction 8. This peak is not observed upon stimulation for the secretory lysosome markers β-hexosaminidase and [3H]serotonin (Fig.2 C). The clear fall in activity from both peaks of the secretory lysosome markers upon stimulation (2C) is not as obvious as the fall in HRP activity, because of the overlap of the peak at fraction 8 with the lighter one of the two secretory lysosome peaks (9Simmen T. Schmidt A. Hunziker W. Beermann F. J. Cell Sci. 1999; 112: 45-53PubMed Google Scholar, 10Blagoveshchenskaya A.D. Hewitt E.W. Cutler D.F. Mol. Biol. Cell. 1999; 10: 3979-3990Crossref PubMed Scopus (60) Google Scholar). The shift of activity to fraction 8 is consecutive over time, HRP activity peaking at fraction 10 for resting and 5 min of stimulation, fraction 9 following 10 min and then to fraction 8 after 25 min of stimulation. This secondary gradient reveals that a large proportion of the residual HRP activity retained in the secretory lysosome peak following 25 min of stimulation in Fig. 2 B has moved to a separate compartment from the markers β-hexosaminidase and [3H]serotonin. In parallel, we quantified release of β-hexosaminidase and [3H]serotonin into the bathing medium. Fig. 2 Eshows that only 3.5% of both β-hexosaminidase and [3H]serotonin is constitutively secreted from resting cells, whereas 47% is released upon 25 min of stimulation. This 47% release is in good agreement with the 45% fall in β-hexosaminidase activity from the subcellular profile of the secretory lysosome in Fig.2 A. We compared the targeting of wt HRP-P-selectin to secretory lysosomes with that of tetra-alanine mutant chimeras of the cytoplasmic sequences KCPL, YGVF, and DPSP and of the deletion mutant 763HRP-P-selectin, which lacks all three motifs (Fig. 3). These three motifs have been implicated in lysosomal, secretory granule, and SLMV targeting (Fig. 1). Rbl cells were transiently transfected with wt,KCPL,YGVF,DPSP, 763HRP-P-selectin and cultured for 3 days. Fractionation was then carried out, and the subcellular profile across the primary sucrose gradient for each chimera is shown in Fig. 4 A. The secretory lysosome fractions are highlighted in gray(14Israels S.J. Gerrard Y.V. Jacques Y.V. McNicol A. Cham B. Nishibori M. Bainton D.F. Blood. 1992; 80: 143-152Crossref PubMed Google Scholar, 15Bonfanti R. Furie B.C. Furie B. Wagner D.D. Blood. 1989; 73: 1109-1112Crossref PubMed Google Scholar, 16McEver R.P. Beckstead J.H. Moore K.L. Marshall-Carlson L. Bainton D.F. J. Clin. Invest. 1989; 84: 92-99Crossref PubMed Scopus (844) Google Scholar, 17Arribas M. Cutler D.F. Traffic. 2000; 1: 783-793Crossref PubMed Scopus (38) Google Scholar, 18Hartwell D.W. Mayadas T.N. Berger G. Frenette P.S. Rayburn H. Hynes R.O. Wagner D.D. J. Cell Biol. 1998; 143: 1129-1141Crossref PubMed Scopus (97) Google Scholar, 19Disdier M. Morrisey J.H. Fugate R.D. Bainton D.F. McEver R.P. Mol. Biol. Cell. 1992; 3: 309-321Crossref PubMed Scopus (128) Google Scholar, 20Modderman P.W. Beuling E.A. Govers L.A.T. Calafat J. Janssen H. Von Dem Borne A.E. Sonnenberg A. Biochem. J. 1998; 336: 153-161Crossref PubMed Scopus (26) Google Scholar). The majority of wt, YGVF,DPSPHRP-P-selectin activity is found in the secretory lysosome peak, all three having very similar profiles. However, much lower levels of HRP activity within the secretory lysosome peak are found with the mutants KCPL and 763HRP-P-selectin, which coincide with a significant accumulation of HRP activity elsewhere on the gradient. The profiles for 763HRP-P-selectin andKCPLHRP-P-selectin differ in that there is a significant shoulder to the major peak for 763HRP-P-selectin such that it covers fractions 6–15, whereas the peak forKCPLHRP-P-selectin is sharper (fractions 10–15). Mutating the sequence KCPL, which is implicated in lysosomal targeting (Fig. 1), to tetra-alanine has a similar effect on targeting to the secretory lysosome as the complete removal of the last 27 residues of the cytoplasmic domain. Because expression ofKCPLHRP-P-selectin in H.Ep.2 cells results in localization in tr
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