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

Shona L Osborne,Tristan P Wallis,Jose L Jimenez,Jeffrey J Gorman,Frederic A Meunier

doi:10.1074/mcp.m600430-mcp200

Shona L Osborne, Tristan P Wallis + Show 3 more

Open Access

https://doi.org/10.1074/mcp.m600430-mcp200

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Abstract

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P2. We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; alpha-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P2 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)P2 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)P2 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)P2 binding, are important for chromaffin granule exocytosis.

Highlights

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells
Syt[7] is endogenously expressed at high levels in chromaffin granules of chromaffin cells compared with PC12 cells, and using a mutational analysis, we demonstrated that the polylysine stretch in Syt[7] C2B is involved in calcium-independent PtdIns(4,5)P2 binding and is important for the inhibitory effect of Syt[7] C2B on chromaffin granule exocytosis
Identification of Chromaffin Granule PtdIns(4,5)P2-binding Proteins—We used a PtdIns(4,5)P2 pulldown strategy combined with mass spectrometry to identify PtdIns(4,5)P2-interacting proteins on chromaffin granules

Summary

EXPERIMENTAL PROCEDURES

Subcellular Fractionation and Immunoisolation—Bovine adrenal medulla were isolated, homogenized, and subjected to differential centrifugation, and purified chromaffin granules were obtained as described previously (27, 28). 50 ␮g of chromaffin and PC12 subcellular fractions were solubilized in Laemmli sample buffer and separated by SDS-PAGE, and proteins were detected by Western blotting with the indicated antibodies. Beads were washed thoroughly in the respective calcium buffers, and associated protein were analyzed by SDS-PAGE and Western blotting using anti-GST antibodies (Sigma). Trypsin digestion was performed by rehydrating dried gel slices in 20 ␮l of 50 ng/␮l trypsin (Roche Applied Science modified sequencing grade) in 10% acetonitrile, 40 mM ammonium bicarbonate and incubating at room temperature for 1 h. Large multilamellar vesicles and associated proteins were collected by centrifugation and washed, and bound proteins were solubilized in Laemmli sample buffer and analyzed by SDS-PAGE and Coomassie Blue staining (n ϭ 3 independent experiments). Catecholamine released was expressed as a percentage of total catecholamine content (n ϭ 8; data shown are representative of three independent experiments)

RESULTS

Synaptotagmin 7

DISCUSSION