Response of an Integral Granule Membrane Protein to Changes in pH
2001; Elsevier BV; Volume: 276; Issue: 32 Linguagem: Inglês
10.1074/jbc.m103936200
ISSN1083-351X
AutoresL. Chastine Bell-Parikh, Betty Eipper, Richard E. Mains,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoA key feature of the regulated secretory pathway in neuroendocrine cells is lumenal pH, which decreases between trans-Golgi network and mature secretory granules. Because peptidylglycine α-amidating monooxygenase (PAM) is one of the few membrane-spanning proteins concentrated in secretory granules and is a known effector of regulated secretion, we examined its sensitivity to pH. Based on antibody binding experiments, the noncatalytic linker regions between the two enzymatic domains of PAM show pH-dependent conformational changes; these changes occur in the presence or absence of a transmembrane domain. Integral membrane PAM-1 solubilized from rat anterior pituitary or from transfected AtT-20 cells aggregates reversibly at pH 5.5 while retaining enzyme activity. Over 35% of the PAM-1 in anterior pituitary extracts aggregates at pH 5.5, whereas only about 5% aggregates at pH 7.5. PAM-1 recovered from secretory granules and endosomes is highly responsive to low pH-induced aggregation, whereas PAM-1 recovered from a light, intracellular recycling compartment is not. Mutagenesis studies indicate that a transmembrane domain is necessary but not sufficient for low pH-induced aggregation and reveal a short lumenal, juxtamembrane segment that also contributes to pH-dependent aggregation. Taken together, these results demonstrate that several properties of membrane PAM serve as indicators of granule pH in neuroendocrine cells. A key feature of the regulated secretory pathway in neuroendocrine cells is lumenal pH, which decreases between trans-Golgi network and mature secretory granules. Because peptidylglycine α-amidating monooxygenase (PAM) is one of the few membrane-spanning proteins concentrated in secretory granules and is a known effector of regulated secretion, we examined its sensitivity to pH. Based on antibody binding experiments, the noncatalytic linker regions between the two enzymatic domains of PAM show pH-dependent conformational changes; these changes occur in the presence or absence of a transmembrane domain. Integral membrane PAM-1 solubilized from rat anterior pituitary or from transfected AtT-20 cells aggregates reversibly at pH 5.5 while retaining enzyme activity. Over 35% of the PAM-1 in anterior pituitary extracts aggregates at pH 5.5, whereas only about 5% aggregates at pH 7.5. PAM-1 recovered from secretory granules and endosomes is highly responsive to low pH-induced aggregation, whereas PAM-1 recovered from a light, intracellular recycling compartment is not. Mutagenesis studies indicate that a transmembrane domain is necessary but not sufficient for low pH-induced aggregation and reveal a short lumenal, juxtamembrane segment that also contributes to pH-dependent aggregation. Taken together, these results demonstrate that several properties of membrane PAM serve as indicators of granule pH in neuroendocrine cells. trans-Golgi network peptidylglycine α-hydroxylating monooxygenase peptidyl-α-hydroxyglycine α-amidating lyase approximately 70-kDa membrane-bound PAL peptidylglycine α-amidating monooxygenase interleukin-2 receptor α chain large dense core vesicle transmembrane domain COOH-terminal domain of PAM, rPAM-1(900–976) antibody monoclonal antibody interleukin-2 receptor α 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid complete serum-free medium 1,4-piperazinediethanesulfonic acid polyacrylamide gel electrophoresis polyvinylidene difluoride 4-morpholineethanesulfonic acid Tac-PAM-PAM PAM-PAM-Tac PAM-Tac-Tac Maturation of bioactive peptides in the nervous and endocrine systems involves a series of post-translational processing steps that occur as precursor proteins and their products traverse the secretory pathway (1Seidah N.G. Chretien M. 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Rev. 1999; 20: 3-21PubMed Google Scholar, 25Chanat E. Huttner W.B. J. Cell Biol. 1991; 115: 1505-1519Crossref PubMed Scopus (387) Google Scholar). Most of the processing of peptides and proteins in the secretory pathway occurs during formation of the immature and mature secretory granules, and acidification appears to play an important role in restricting proteolytic processing to late secretory compartments. Proopiomelanocortin, the precursor to several bioactive peptides, is cleaved and stored in large dense core vesicles (LDCVs) of select endocrine cells and neurons (6Mains R.E. Eipper B.A. Siegel G.R. Agranoff B.W. Albers R.W. Fisher S.K. Uhler M.D. Basic Neurochemistry. Lippincott-Raven, Philadelphia, PA1999: 363-382Google Scholar). Acidification to approximately pH 6.0 promotes proopiomelanocortin processing (14Schmidt W.K. Moore H.P.H. Mol. Biol. Cell. 1995; 6: 1271-1285Crossref PubMed Scopus (61) Google Scholar). This is consistent with evidence demonstrating that peptide processing enzymes targeted to secretory vesicles require acidic conditions for activation. Furin and prohormone convertases such as prohormone convertase 2 require low pH for activation (4Muller L. Lindberg I. Prog. Nucleic Acid Res. Mol. Biol. 1999; 63: 69-108Crossref PubMed Scopus (125) Google Scholar, 26Lindberg I. van den Hurk W.H. Bui C. Batie C.J. Biochemistry. 1995; 34: 5486-5493Crossref PubMed Scopus (76) Google Scholar, 27Anderson E.D. VanSlyke J.K. Thulin C.D. Jean F. Thomas G. EMBO J. 1997; 16: 1508-1518Crossref PubMed Scopus (198) Google Scholar). Furin, for example, must be exposed to a low enough pH to release its cleaved but tightly bound prosequence (27Anderson E.D. VanSlyke J.K. Thulin C.D. Jean F. Thomas G. EMBO J. 1997; 16: 1508-1518Crossref PubMed Scopus (198) Google Scholar). It is therefore evident that a number of secretory granule proteins exhibit physiologically relevant sensitivities to the changes in pH that occur in the secretory pathway. PAM is found in nearly all cells with LDCVs and is one of the few peptide processing enzymes that spans the vesicle membrane (28Eipper B.A. Stoffers D.A. Mains R.E. Annu. Rev. Neurosci. 1992; 15: 57-85Crossref PubMed Scopus (559) Google Scholar). PAM, a bifunctional enzyme, first catalyzes the hydroxylation of glycine-extended peptides through its hydroxylation domain (PHM) and subsequently catalyzes α-amidation through its lyase domain (PAL) (2Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (373) Google Scholar,28Eipper B.A. Stoffers D.A. Mains R.E. Annu. Rev. Neurosci. 1992; 15: 57-85Crossref PubMed Scopus (559) Google Scholar). Like other peptide processing enzymes, PAM is most active at low pH. In addition to its enzymatic role, a significant body of evidence suggests a role for integral membrane PAM in mediating regulated secretion through its effects on cytoskeletal organization; these effects on regulated secretion and the cytoskeleton are not observed when soluble PAM constructs are expressed (29Ciccotosto G.D. Schiller M.R. Eipper B.A. Mains R.E. J. Cell Biol. 1999; 144: 459-471Crossref PubMed Scopus (54) Google Scholar, 30Mains R.E. Alam M.R. Johnson R.C. Darlington D.N. Back N. Hand T.A. Eipper B.A. J. Biol. Chem. 1999; 274: 2929-2937Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 31Alam M.R. Steveson T.C. Johnson R.C. Back N. Abraham B. Mains R.E. Eipper B.A. Mol. Biol. Cell. 2001; 12: 629-644Crossref PubMed Scopus (28) Google Scholar). The PAM cytosolic domain interacts with and affects the localization of several cytoskeletal-associated cytosolic proteins (30Mains R.E. Alam M.R. Johnson R.C. Darlington D.N. Back N. Hand T.A. Eipper B.A. J. Biol. Chem. 1999; 274: 2929-2937Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 31Alam M.R. Steveson T.C. Johnson R.C. Back N. Abraham B. Mains R.E. Eipper B.A. Mol. Biol. Cell. 2001; 12: 629-644Crossref PubMed Scopus (28) Google Scholar, 32Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 33Alam M.R. Johnson R.C. Darlington D.N. Hand T.A. Mains R.E. Eipper B.A. J. Biol. Chem. 1997; 272: 12667-12675Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Expression of PAM-1 in AtT-20 cells leads to rearrangement of both actin and intermediate filament components of the cytoskeleton and results in a significant decrease in regulated secretion (29Ciccotosto G.D. Schiller M.R. Eipper B.A. Mains R.E. J. Cell Biol. 1999; 144: 459-471Crossref PubMed Scopus (54) Google Scholar). Because regulated secretion is affected by molecules in and on the LDCV, it stands to reason that there is some level of communication between the lumen of the LDCV and the cytosol. Lumenal proteins such as carboxypeptidase E and the chromogranins display pH-dependent protein-protein interactions, conformational changes, and membrane association (17Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar, 18Yoo S.H. J. Biol. Chem. 1996; 271: 1558-1565Abstract Full Text Full Text PDF PubMed Google Scholar, 21Song L. Fricker L.D. J. Biol. Chem. 1995; 270: 7963-7967Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 22Rindler M.J. J. Biol. Chem. 1998; 273: 31180-31185Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 34Cool D.R. Normant E. Shen F.S. Chen H.C. Pannell L. Zhang Y. Loh Y.P. Cell. 1997; 88: 73-83Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Although some of these proteins are membrane-associated, none actually spans the vesicle membrane. Because PAM was known to exhibit pH-sensitive changes in catalytic rate, we explored the possibility that PAM, like other proteins in the regulated secretory pathway, exhibited additional pH-sensitive properties. In this study we explored the possibility that PAM could serve in a signal transduction system linking the lumenal environment to cytosolic factors that regulate the cytoskeleton or other cellular functions. We employed subcellular fractionation, sucrose gradient sedimentation, and immunoprecipitation to explore the sensitivity of PAM to changes in pH. Our results demonstrate that PAM is highly responsive to the pH changes that characterize granule maturation and support the possibility that PAM acts as a pH sensor within LDCVs. AtT-20 cells expressing PAM-1, PAM-1/899, PAM-2, PAM-3, TPP (35Milgram S.L. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 17526-17535Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and myc-TMD-CD (previously called kp-myc-CD) (36Bruzzaniti A. Marx R. Mains R.E. J. Biol. Chem. 1999; 274: 24703-24713Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) were described previously. HEK293 cells expressing PAM-1 were described (37Tausk F.A. Milgram S.L. Mains R.E. Eipper B.A. Mol. Endocrinol. 1992; 6: 2185-2196PubMed Google Scholar). Antisera to PAM were described previously: mouse monoclonal antibody specific for the COOH-terminal domain (6E6; rPAM-1(898–976); CD mAb) (38Milgram S.L. Kho S.T. Martin G.V. Mains R.E. Eipper B.A. J. Cell Sci. 1997; 110: 695-706Crossref PubMed Google Scholar); rabbit polyclonal antisera raised against PHM (Abs 1761 and 1764; rPAM-1(37–382)), PAL (Ab 471), Exon 16 (also called Exon A) (Ab 629), and the COOH-terminal domain (Ab 571; CD polyclonal Ab) (30Mains R.E. Alam M.R. Johnson R.C. Darlington D.N. Back N. Hand T.A. Eipper B.A. J. Biol. Chem. 1999; 274: 2929-2937Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 39Yun H.-Y. Johnson R.C. Mains R.E. Eipper B.A. Arch. Biochem. Biophys. 1993; 301: 77-84Crossref PubMed Scopus (34) Google Scholar, 40Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: 717-728Crossref PubMed Scopus (103) Google Scholar). Although Ab 475 was generated to rPAM-1(37–382), it fails to detect rPAM-1(37–369) and will be referred to as an Exon 15 (rPAM-1(369–392)) antibody (41Eipper B.A. Quon A.S.W. Mains R.E. Boswell J.S. Blackburn N.J. Biochemistry. 1995; 34: 2857-2865Crossref PubMed Scopus (102) Google Scholar). Antibody against the COOH terminus of Tac (IL-2Rα, C-20) was from Santa Cruz Biotechnology. The expression vectors pCIS.PPT, pCIS.PTT, and pCDM.Tac were generously provided by Dr. Sharon Milgram (University of North Carolina, Chapel Hill, NC). Transfected cell lines were plated onto tissue culture dishes or slides pretreated with poly-lysine and NuSerum and kept in growth medium (Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 10% fetal calf serum (HyClone, Logan, UT), 10% NuSerum (Collaborative Research, Bedford, MA), and antibiotics) containing 0.5 mg/ml G418. Triton X-100, CHAPS, Sarkosyl, and octyl-β-glucopyranoside were from Calbiochem. Complete serum-free medium (CSFM) is Dulbecco's modified Eagle's medium/Ham's F-12 medium containing insulin and transferrin; CSFM-Air has HEPES buffer substituted for the bicarbonate (42Ratovitski E.A. Alam M.R. Quick R.A. McMillan A. Bao C. Hand T.A. Johnson R.C. Mains R.E. Eipper B.A. Lowenstein C.J. J. Biol. Chem. 1999; 274: 993-999Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To express soluble, cytosolic CD, pBS.KrPAM-1 was used to amplify the appropriate fragment with 5′ SalI and 3′ BamHI restriction sites (43Husten E.J. Eipper B.A. Arch. Biochem. Biophys. 1994; 312: 487-492Crossref PubMed Scopus (29) Google Scholar). This fragment was inserted into SalI-BamHI cut pRK5 (33Alam M.R. Johnson R.C. Darlington D.N. Hand T.A. Mains R.E. Eipper B.A. J. Biol. Chem. 1997; 272: 12667-12675Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) to produce myc-Gly5-Ser-Thr-PAM (891). The construct was verified by sequencing. For expression ofmyc-CD (pRK.myc-CD), PAM/Tac chimeras (pCIS.PTT or pCIS.PPT), and Tac (pCDM.Tac), AtT-20 cells were co-transfected with expression vector (20 µg) and pCI.neo (5 µg) using Lipofectin (Life Technologies, Inc.). Stably transfected lines were selected and maintained in growth medium plus 0.5 mg/ml G418. For each cell line, two clones with similar expression levels were analyzed (35Milgram S.L. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 17526-17535Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Stably transfected AtT-20 or HEK293 cells were kept for 2 days in growth medium. The cells of one confluent well of a 12-well dish were preincubated in 500 µl of CSFM-Air for 5 min at 37 °C. Warm medium was replaced with ice-cold co-immunoprecipitation buffer (20 mm PIPES, 2 mm Na2EDTA, 50 mm NaF, 10 mm Na4P2O7, and 1 mm Na3VO4, pH 7.5) with protease inhibitors (30 µg/ml phenylmethylsulfonyl fluoride, 16 µg/ml benzamidine, 2 µg/ml leupeptin, 10 µg/ml lima bean trypsin inhibitor) and 1% Triton X-100 (unless otherwise specified) (250 µl), and the cells were incubated on ice for 15 min (42Ratovitski E.A. Alam M.R. Quick R.A. McMillan A. Bao C. Hand T.A. Johnson R.C. Mains R.E. Eipper B.A. Lowenstein C.J. J. Biol. Chem. 1999; 274: 993-999Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The cells were subsequently scraped from the dish, and the suspension (representing total PAM) was centrifuged for 20 min at 250,000 ×g at 4 °C. For extraction of PAM-1 from rat pituitaries, two whole adult, male, Harlan Sprague-Dawley rat pituitaries were homogenized in 500 µl of co-immunoprecipitation buffer, pH 7.5, with protease inhibitors and 1% Triton X-100 using a motor-driven Potter-Elvehjem homogenizer with a Teflon pestle at 4 °C. Homogenate was centrifuged for 20 min at 250,000 × g at 4 °C, and the supernatant (100 µl/gradient) was used for sucrose gradient sedimentation studies. Detergent-soluble PAM-1 (S) was recovered in the supernatant and used to perform most of the experiments described. Detergent-insoluble PAM (I) was recovered by suspending the pellet in 250 µl of co-immunoprecipitation buffer, pH 7.5. For comparison of extraction efficiencies, equal volumes (20 µl) of the soluble (S) and insoluble (I) fractions were analyzed, along with 20 µl of cell extract (T). Following SDS-PAGE and transfer to PVDF membranes, PAM was visualized using Exon 16 Ab 629 and chemiluminescence;ScionImage (NIH) was used to quantify images. Each of two confluent wells of a 12-well dish of AtT-20 cells expressing PAM-2 or PAM-1/899 was infected with PAM-1 adenovirus (44Marx R. El Meskini R. Johns D.C. Mains R.E. J. Neurosci. 1999; 19: 8300-8311Crossref PubMed Google Scholar) and extracted into 150 µl of ice-cold co-immunoprecipitation buffer following a brief rinse in warm medium as described above. Solubilized protein from the two wells was pooled, and 25 µl was incubated in 500 µl of co-immunoprecipitation buffer titrated to pH 5.5 or to 7.5 and containing 10 µl of the specified polyclonal antibody. Antibody binding proceeded for 2 h at 4 °C. Protein A-agarose beads (Sigma) were preblocked with 2.0 mg/ml bovine serum albumin in phosphate-buffered saline and then equilibrated with pH 5.5 or 7.5 co-immunoprecipitation buffer. Following antibody binding, the samples were centrifuged for 20 min at 5000 rpm at 4 °C in a tabletop centrifuge, and the supernatant (200 µl for each pH) was incubated with pretreated Protein A beads (60 µl of a 33% slurry) for 1 h at 4 °C. The beads were pelleted and washed twice with co-immunoprecipitation buffer of the same pH. Proteins eluted by boiling for 5 min in Laemmli sample buffer (1% SDS (w/v), 8m urea, 5% 2-mercaptoethanol (v/v), 50 mmTris-HCl, pH 6.8) were fractionated by SDS-PAGE. Western blots were visualized using PAM CD mAb 6E6 ot the PHM Ab. Multiple aliquots of an AtT-20 PAM-1 cell extract prepared as described above were fractionated by SDS-PAGE. Following transfer, the PVDF membrane was cut into strips of two lanes each for antibody binding so that each antibody and pH was tested in duplicate. All membranes were initially blocked for 45 min with 5% milk in 50 mm Tris-HCl, pH 7.5, 150 mmNaCl, 0.1% Tween 20 (TTBS) and rinsed with TTBS at pH 7.5. Membrane strips were incubated for 2 h with Exon 16 Ab, Exon 15 Ab, or PAM CD mAb diluted into either TTBS, pH 7.5 or 50 mm Na-MES, 0.1% Tween 20, pH 5.5. Subsequent rinses and secondary antibody binding used TTBS, pH 7.5. Protein was visualized and quantified as described above. AtT-20 cells expressing PAM-1/899 were plated onto poly-lysine treated 4-well glass slides and grown for at least 36 h. Prior to antibody binding, cells were incubated for 20 min at 37 °C in CSFM-Air. Polyclonal antibodies to Exon 15 or PHM were diluted 1:50 into CSFM-Air with the pH adjusted to 7.3 or 5.7 using NaOH. The cells were incubated with antibody for 1 h at 4 °C to permit binding. This incubation was followed by a brief rinse with 4 °C CSFM-Air medium of the same pH. The cells were then fixed by incubation in ice-cold 100% methanol for 10 min. Bound antibody was visualized using fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody followed by fluorescence microscopy. Sucrose (5%, 20%, and 50% (w/v)) was dissolved in co-immunoprecipitation buffer adjusted to pH 5.5 or pH 7.5 (42Ratovitski E.A. Alam M.R. Quick R.A. McMillan A. Bao C. Hand T.A. Johnson R.C. Mains R.E. Eipper B.A. Lowenstein C.J. J. Biol. Chem. 1999; 274: 993-999Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 45Bennett M.K. Calakos N. Kreiner T. Scheller R.H. J. Cell Biol. 1992; 116: 761-775Crossref PubMed Scopus (181) Google Scholar) without detergent (unless otherwise indicated). Gradients consisted of a 2.0-ml linear 5–20% sucrose gradient with a 50% sucrose "pad" (169 µl) beneath. Molecular mass markers (50–250 µg of apoferritin, catalase, bovine serum albumin, ovalbumin, and cytochrome c) dissolved in co-immunoprecipitation buffer were used as internal or external standards. Samples (up to 200 µl) loaded on top of the sucrose gradients were centrifuged for 5 h at 4 °C, 50,000 rpm (214,000 × g) in a Ti-55 swinging bucket rotor in a TL100 centrifuge (Beckman). Fractions (169 µl) from each gradient were collected from the top down. Particulate matter that remained on the bottom of the tube was recovered by adding Laemmli sample buffer (169 µl) and heating for 10 min at 37 °C or by adding immunoprecipitation buffer (169 µl), pH 7.5, with detergent (as specified). This final fraction was collected and labeled "Particulate." Gradient fractions were analyzed on 10% polyacrylamide, 0.25%N,N′-methylene-bisacrylamide/SDS gels. Following transfer to PVDF membranes (Millipore Corp.), PAM proteins were visualized with Exon A antibody (1:1000) or CD mAb (1:20) using theAmersham Pharmacia Biotech ECL kit. Tac (IL-2Rα) was detected using a COOH-terminal rabbit polyclonal antibody (1:1000; IL-2Rα (C-20) sc-664, Santa Cruz Biotechnology). Total protein was visualized using Coomassie Brilliant Blue R-250. Pituitaries from 15–20 adult male Harlan Sprague-Dawley rats (150–200 g) (Charles River, Wilmington, MA) were homogenized using a motor-driven PotterElvehjem homogenizer with a Teflon pestle at 4 °C in 10 volumes (w/v) of 0.32 m sucrose, 10 mmTris-HCl, pH 7.4, containing protease inhibitors (46Oyarce A.M. Eipper B.A. J. Cell Sci. 1995; 108: 287-297Crossref PubMed Google Scholar, 47El Meskini R. Mains R.E. Eipper B.A. Endocrinology. 2000; 141: 3020-3034Crossref PubMed Scopus (26) Google Scholar). Differential centrifugation yielded pellets enriched in membranous structures, secretory granules, and endosomes (referred to as P2, P3 and P4, respectively) (46Oyarce A.M. Eipper B.A. J. Cell Sci. 1995; 108: 287-297Crossref PubMed Google Scholar). Pellets were homogenized in 200 µl of homogenization buffer at 4 °C and loaded onto discontinuous sucrose gradients to further separate organelles (46Oyarce A.M. Eipper B.A. J. Cell Sci. 1995; 108: 287-297Crossref PubMed Google Scholar). The appropriate fractions from each gradient were pooled to obtain preparations enriched in light vesicles (P2, Pool A), secretory granules (P3, Pool C), and endosomes (P4, Pool B) (46Oyarce A.M. Eipper B.A. J. Cell Sci. 1995; 108: 287-297Crossref PubMed Google Scholar). These pooled fractions (500 µl) were diluted to 3.0 ml with homogenization buffer so that organelles could be pelleted by centrifugation for 15 min at 350,000 ×g. Each of these pellets was then resuspended in 400 µl of co-immunoprecipitation buffer, pH 7.5, for solubilization of membranes and extraction of PAM. Finally, 175-µl aliquots of each resuspended fraction were loaded onto each of two separate 2.0-ml 5–20% linear sucrose gradients, one at pH 5.5 and one at pH 7.5. These gradients were centrifuged, separated, and analyzed as described above. To investigate the possibility of pH-dependent structural changes in PAM, extracts of AtT-20 cells expressing full-length PAM-1 were immunoprecipitated under pH conditions mimicking those in the Golgi or in mature secretory granules; antibodies to discrete domains of the PAM protein were used (Fig. 1 A). Most of the PAM antibodies were comparable in their ability to immunoprecipitate PAM-1 at pH 5.5 and 7.5 (Fig. 1 B). Two of the antibodies examined (to Exon 15 and Exon 16) successfully immunoprecipitated PAM-1 from extracts at pH 7.5, but their capacities for immunoprecipitating PAM-1 at pH 5.5 were greatly diminished. The inability of these antibodies to recognize PAM-1 at pH 5.5 could reflect the properties of PAM-1 or properties of the antibodies. To distinguish between these possibilities, we asked whether the Exon 16 antibody and/or a control antibody (CD mAb) exhibited pH-sensitive binding to PAM-1 that had been reduced and denatured, fractionated by SDS-PAGE, and transferred to PVDF membranes (Fig. 1 C). Neither of these antibodies showed a pH-dependent interaction with denatured PAM. Thus the inability of the Exon 16 antibody to recognize native PAM-1 at pH 5.5 reflects a pH-dependent change in PAM-1 rather than a pH sensitivity of the antibody. Similarly, the Exon 15 antibody detected PAM-1 equally well at pH 7.5 and at pH 5.5 on Western blots (not shown). Alternative splicing generates isoforms of PAM lacking the transmembrane domain and/or Exon 16 (Fig.2 A). To delineate the contribution of these domains to the observed pH sensitivity, natural isoforms lacking these domains were exposed to antibody at pH 5.5 and at pH 7.5 (Fig. 2 B). The ability of the Exon 15 antibody to recognize both PAM-2 (which lacks Exon 16) and PAM-3 (which lacks both the transmembrane domain and Exon 16) was greatly reduced at pH 5.5. In contrast, the ability of the CD polyclonal antibody to immunoprecipitate PAM-2 and PAM-3 was not pH-dependent (Fig. 2 B). From these results, we conclude that sequences contained within Exons 15 and 16 exhibit pH-dependent changes in conformation. Additionally, based on the response of PAM-3, it appears that the transmembrane domain is not required for this pH-responsive change in antibody reactivity. Immunoprecipitation of membrane PAM requires its solubilization. We carried out additional antibody binding experiments with live cells to determine whether PAM embedded in the lipid bilayer still exhibited pH-sensitive epitope masking. AtT-20 cells overexpressing PAM-1/899, a membrane protein with a truncated cytosolic domain but possessing t
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