Identification of Secretory Granule Phosphatidylinositol 4,5-Bisphosphate-interacting Proteins Using an Affinity Pulldown Strategy

Abstract

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P 2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P 2 . We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; ␣-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P 2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P 2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P 2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P 2 binding, are important for chromaffin granule exocytosis. Molecular & Cellular Proteomics 6:1158 -1169, 2007.Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 plays a critical role in neurosecretory cells for both the exocytosis and endocytosis of secretory vesicles (1-3). The PtdIns(4,5)P 2 required for secretion is synthesized on the plasma membrane (4), and the binding of vesicle-associated proteins to PtdIns(4,5)P 2 in trans is thought to be important for bringing the vesicle and plasma membranes together prior to exocytosis to ensure rapid and efficient fusion upon calcium influx (5). Putative PtdIns(4,5)P 2 effectors include synaptotagmin 1 (Syt1) (6, 7), calcium-activated protein for secretion (CAPS) (8, 9), and rabphilin (10).CAPS binds to PtdIns(4,5)P 2 and is a potential PtdIns(4,5)P 2 effector for priming of secretory granule exocytosis (9). However, recent work on the CAPS1 knock-out mouse highlighted an involvement of CAPS1 in the refilling or storage of catecholamine in mature secretory granules therefore suggesting an alternative role for CAPS (11).Syts play a fundamental role in triggering membrane fusion in a variety of systems, and much work has focused on Syt1 as the main candidate for calcium sensor in exocytosis (12)(13)(14). Syt1 binds to PtdIns(4,5)P 2 , and this interaction is believed to play a critical role in bridging the secretory vesicle and plasma membranes during exocytosis (5, 6). However, multiple Syt isoforms are expressed in the central nervous system and neurosecretory cells, suggesting that different isoforms ma...

PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells

“…For example, there is evidence that phosphatidylinositol 4,5-bisphosphate plays a role in coordinating both exocytosis and endocytosis at the synapse (10,11,13,47), whereas PtdIns(3)P, a lipid best characterized for its role in early endosome function (48), has also been shown to be involved in lysophosphatidic acid-stimulated cell migration (49), ATP-dependent priming in neurosecretory cells (12), and translocation of GLUT4-containing vesicles to the plasma membrane (50,51). An important point was therefore to investigate whether PtdIns(3,5)P 2 is produced upon stimulation of exocytosis.…”

“…Much work has been directed toward identifying the proteins involved in and regulating the exocytic process. These include members of the SNARE family of proteins, synaptotagmins (1,2,4,5,13), and a host of regulatory proteins, such as Munc18a (6,7). In addition to the critical role played by membrane proteins, there is an increasing amount of literature pointing to the importance of lipids, in particular phosphoinositides, in the regulation of membrane trafficking events, including regulated exocytosis (8 -13).…”

Ca2+-regulated Pool of Phosphatidylinositol-3-phosphate Produced by Phosphatidylinositol 3-Kinase C2α on Neurosecretory Vesicles

“…The purity of our preparation was established previously (Bon et al, 1990;Vitale et al, 1996;Meunier et al, 2005;Osborne et al, 2007), and Western blot analysis demonstrated that no early endosomal contamination could be detected (Meunier et al, 2005). Chromaffin granules were first incubated in various conditions including ATP, cytosol extract.…”

“…Chromaffin granules were prepared as described previously (Bon et al, 1990;Vitale et al, 1996;Meunier et al, 2005;Osborne et al, 2007). Briefly, bovine adrenal medulla were taken and finely chopped in 0.32 M sucrose (10 mM Tris, 1 mM EGTA, pH 7.4, and protease inhibitor [Roche Products, Dee Why, NSW, Australia]).…”

“…Purified chromaffin granules were prepared from bovine adrenal medulla as described previously (Bon et al, 1990;Vitale et al, 1996;Meunier et al, 2005;Osborne et al, 2007). We incubated 125 g of granules per assay with 0.5 g of 2xFYVE-GST for 30 min at 37°C in KGEP buffer containing 2 mM free Mg 2ϩ and 2 mM ATP and the indicated concentrations of free calcium calculated using the WEBMAXC program (Patton et al, 2004).…”