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Identification of Secretory Granule Phosphatidylinositol 4,5-Bisphosphate- Interacting Proteins Using an Affinity Pulldown Strategy*□S

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Identification of Secretory Granule Phosphatidylinositol 4,5-Bisphosphate- Interacting Proteins Using an Affinity Pulldown Strategy*□S Research Identification of Secretory Granule Phosphatidylinositol 4,5-Bisphosphate- interacting Proteins Using an Affinity Pulldown Strategy*□S Shona L. Osborne‡§, Tristan P. Wallis¶ʈ, Jose L. Jimenez**, Jeffrey J. Gorman¶ʈ, and Frederic A. Meunier‡§‡‡ Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) syn- required for secretion is synthesized on the plasma mem- thesis is required for calcium-dependent exocytosis in neu- brane (4), and the binding of vesicle-associated proteins to rosecretory cells. We developed a PtdIns(4,5)P2 bead pull- PtdIns(4,5)P2 in trans is thought to be important for bringing down strategy combined with subcellular fractionation to the vesicle and plasma membranes together prior to exocy- Downloaded from identify endogenous chromaffin granule proteins that inter- tosis to ensure rapid and efficient fusion upon calcium influx act with PtdIns(4,5)P . We identified two synaptotagmin iso- 2 (5). Putative PtdIns(4,5)P effectors include synaptotagmin 1 forms, synaptotagmins 1 and 7; spectrin; ␣-adaptin; and 2 (Syt1) (6, 7), calcium-activated protein for secretion (CAPS) (8, synaptotagmin-like protein 4 (granuphilin) by mass spec- 9), and rabphilin (10). trometry and Western blotting. The interaction between https://www.mcponline.org CAPS binds to PtdIns(4,5)P2 and is a potential PtdIns(4,5)P2 synaptotagmin 7 and PtdIns(4,5)P2 and its functional rele- vance was investigated. The 45-kDa isoform of synaptotag- effector for priming of secretory granule exocytosis (9). How- min 7 was found to be highly expressed in adrenal chromaf- ever, recent work on the CAPS1 knock-out mouse highlighted fin cells compared with PC12 cells and to mainly localize to an involvement of CAPS1 in the refilling or storage of cate- secretory granules by subcellular fractionation, immunoiso- cholamine in mature secretory granules therefore suggesting lation, and immunocytochemistry. We demonstrated that an alternative role for CAPS (11). synaptotagmin 7 binds PtdIns(4,5)P2 via the C2B domain in Syts play a fundamental role in triggering membrane fusion the absence of calcium and via both the C2A and C2B in a variety of systems, and much work has focused on Syt1 at UQ Library on April 27, 2020 domains in the presence of calcium. We mutated the poly- as the main candidate for calcium sensor in exocytosis (12– lysine stretch in synaptotagmin 7 C2B and demonstrated 14). Syt1 binds to PtdIns(4,5)P , and this interaction is be- that this mutant domain lacks the calcium-independent 2 lieved to play a critical role in bridging the secretory vesicle PtdIns(4,5)P2 binding. Synaptotagmin 7 C2B domain inhib- ited catecholamine release from digitonin-permeabi- and plasma membranes during exocytosis (5, 6). However, lized chromaffin cells, and this inhibition was abrogated multiple Syt isoforms are expressed in the central nervous sys- with the C2B polylysine mutant. These data indicate that tem and neurosecretory cells, suggesting that different isoforms synaptotagmin 7 C2B-effector interactions, which occur may be involved in modulating exocytosis through mechanisms via the polylysine stretch, including calcium-indepen- such as homo- and hetero-oligomerization (7, 15, 16). dent PtdIns(4,5)P2 binding, are important for chromaffin Among the Syts capable of modulating secretory granule granule exocytosis. Molecular & Cellular Proteomics exocytosis, Syt7 has generated controversy (13, 17). Syt7 was 6:1158–1169, 2007. initially postulated to be a plasma membrane calcium sensor for exocytosis (18), although more recent studies have local- ized it to secretory granules in PC12 cells (17, 19). However, 1 Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) plays to date, Syt7 localization studies in PC12 cells have been a critical role in neurosecretory cells for both the exocytosis carried out using overexpression studies. Furthermore RNA and endocytosis of secretory vesicles (1–3). The PtdIns(4,5)P2 interference knockdown of endogenous Syt7 in PC12 cells has varying effects on secretion (17, 19) so the physiological From the ‡Molecular Dynamics of Synaptic Function Laboratory, significance of Syt7 in PC12 cells remains unclear. Likewise School of Biomedical Sciences and ¶Institute for Molecular Bio- science, University of Queensland, St. Lucia, Queensland 4072, Aus- tralia and **Wellcome Trust Sanger Institute, Hinxton, Cambridge 4,5-bisphosphate; CAPS, calcium-activated protein for secretion; CB10 1SA, United Kingdom CgA, chromogranin A; IPP, inositol polyphosphate; PtdIns, phospha- Received, November 14, 2006, and in revised form, March 15, 2007 tidylinositol; Syt, synaptotagmin; PtdIns(3,5)P2, phosphatidylinositol Published, MCP Papers in Press, April 20, 2007, DOI 10.1074/ 3,5-bisphosphate; VAMP, vesicle-associated membrane protein; mcp.M600430-MCP200 ACTH, adrenocorticotropin; TBST, TBS with Tween 20; PH, pleckstrin 1 The abbreviations used are: PtdIns(4,5)P2, phosphatidylinositol homology; Syx/S25, syntaxin/SNAP-25. 1158 Molecular & Cellular Proteomics 6.7 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Secretory Granule PtdIns(4,5)P2-Protein Interactions the involvement of Syt7 in neurotransmitter release in the central mM DTT) containing Complete protease inhibitor mixture (Roche Ap- nervous system remains to be determined. Syt7 knock-out mice plied Science), phosphatase inhibitor mixtures I and II (Sigma), and do not have any obvious neurological defects (20); however, 0.5% Nonidet P-40 at 2 mg/ml. Solubilized material was precleared by centrifugation for 15 min at 13,000 rpm at 4 °C and incubated for there is evidence that Syt7 splice variants can regulate the 3 h at 4 °C with either 1 nmol of biotinylated C8-PtdIns(4,5)P2 (Cell- speed of synaptic vesicle recycling (21). Unlike Syt1, Syt7 is Signals Inc., Columbus, OH) (29) prebound to UltraLink Plus NeutrA- widely expressed, and there is compelling evidence that in vidin beads (Pierce) or beads alone. Beads were washed extensively non-neuronal cells Syt7 plays a role in lysosomal exocytosis, in Buffer A containing 0.5% Nonidet P-40 and then resuspended in ␤ membrane resealing, and wound healing (20, 22–26). Laemmli sample buffer containing -mercaptoethanol. For calcium binding studies, chromaffin granules were solubilized in the presence In view of the critical role of PtdIns(4,5)P in exocytosis, an 2 of4mM EGTA or 4 mM EGTA with 100 ␮M free calcium, and beads important issue is to identify possible PtdIns(4,5)P2 effectors were washed in the respective calcium buffers. Calcium concentra- involved in neurosecretion and to examine their role(s) in tions were calculated using the WEBMAXC program (66). Proteins associated with the pulldowns were analyzed by Western blotting exocytosis. To address this we used a PtdIns(4,5)P2 binding ␣ ␤ ␣ pulldown strategy combined with mass spectrometry to iden- with antibodies raised against / spectrin (Chemicon), -adaptin (BD Biosciences), granuphilin (30), synaptotagmin 1 (31), chromogranin A, tify PtdIns(4,5)P -interacting proteins. Adrenal chromaffin 2 synaptotagmin 7, and VAMP2 (Synaptic Systems). Recombinant pro- cells are a widely used and well characterized system for teins were also assayed for binding to the PtdIns(4,5)P2 beads: 100 ng studying regulated exocytosis. Highly purified secretory of eluted GST fusion protein was incubated with PtdIns(4,5)P2 beads (chromaffin) granules from bovine adrenal chromaffin cells as above. Beads were washed thoroughly in the respective calcium Downloaded from were therefore used as a source of protein to minimize the buffers, and associated protein were analyzed by SDS-PAGE and number of unrelated PtdIns(4,5)P -binding proteins. As ex- Western blotting using anti-GST antibodies (Sigma). 2 In-gel Digestions—Gel slices were excised from gels and destained pected, we positively identified Syt1 as a chromaffin granule with two washes in 200 ␮l of 50% acetonitrile, 200 mM ammonium PtdIns(4,5)P2-interacting protein by mass spectrometry. Inter- bicarbonate at 37 °C for 45 min. Destained gel pieces were dried in a estingly spectrin, ␣-adaptin, Syt7, and synaptotagmin-like centrifugal concentrator and reduced by rehydrating with 100 ␮lof20 https://www.mcponline.org protein 4 (granuphilin) were also identified, and these identi- mM DTT, 25 mM ammonium bicarbonate followed by incubation at 65 °C under N . After discarding the supernatant the reduced gel fications were confirmed by Western blotting. Syt7 is endog- 2 slices were alkylated by incubating in 100 ␮lof50mM iodoacetamide, enously expressed at high levels in chromaffin granules of 25 mM ammonium bicarbonate at 37 °C for 40 min in the dark under chromaffin cells compared with PC12 cells, and using a mu- ␮ N2. Gel slices were washed with three changes of 200 lof25mM tational analysis, we demonstrated that the polylysine stretch ammonium bicarbonate at 37 °C for 15 min and redried. Trypsin ␮ in Syt7 C2B is involved in calcium-independent PtdIns(4,5)P2 digestion was performed by rehydrating dried gel slices in 20 lof50 ng/␮l trypsin (Roche Applied Science modified sequencing grade) in binding and is important for the inhibitory effect of Syt7 C2B at UQ Library on April 27, 2020 10% acetonitrile, 40 mM ammonium bicarbonate and incubating at on chromaffin granule exocytosis. room temperature for 1 h. An additional 50 ␮l of 10% acetonitrile, 40 mM ammonium bicarbonate was added, and the incubation continued EXPERIMENTAL PROCEDURES for 18 h at 37 °C. Supernatant containing tryptic peptides was re- Subcellular Fractionation and Immunoisolation—Bovine adrenal moved to a fresh tube, and peptides were extracted from the gel slice ␮ medulla were isolated, homogenized, and subjected to differential by three washes in 50 l of 0.1% TFA at 37 °C for 45 min. Extracts centrifugation, and purified chromaffin granules were obtained as were pooled and concentrated using ZipTips (Millipore) largely ac- ␮ described previously (27, 28). Protein concentrations were deter- cording to manufacturer’s instructions with peptides eluted in 10 lof mined by Bradford protein assay (Bio-Rad).
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