An N-terminal amphipathic helix binds phosphoinositides and enhances Kalirin Sec14 domain-mediated membrane interactions [Neurobiology]

April 10th, 2015 by Miller, M. B., Vishwanatha, K. S., Mains, R. E., Eipper, B. A.

Previous studies revealed an essential role for the lipid-binding Sec14 domain of Kalirin (KalSec14), but its mechanism of action is not well understood. Since alternative promoter usage appends unique N-terminal peptides to the KalSec14 domain, we used biophysical, biochemical and cell biological approaches to examine the two major products, bKalSec14 and cKalSec14. Promoter B encodes a charged, unstructured peptide, while promoter C encodes an amphipathic helix (Kal-C-helix). Both bKalSec14 and cKalSec14 interacted with lipids in phosphatidylinositol phosphate (PIP) strip and liposome flotation assays, with significantly greater binding by cKalSec14 in both assays. Disruption of the hydrophobic face of the Kal-C-helix in cKalSec14KKED eliminated its increased liposome binding. While cKalSec14 showed significantly reduced binding to liposomes lacking PIPs or cholesterol, liposome binding by bKalSec14 and cKalSec14KKED was not affected. When expressed in AtT-20 cells, bKalSec14-GFP was diffusely localized, while cKalSec14-GFP localized to the trans-Golgi Network (TGN) and secretory granules. The amphipathic C-helix was sufficient for this localization. When AtT-20 cells were treated with a cell-permeant derivative of the Kal-C-helix (Kal-C-helix-Arg9), we observed increased secretion of a product stored in mature secretory granules, with no effect on basal secretion; a cell-permeant control peptide (Kal-C-helixKKED-Arg9) did not have this effect. Through its ability to control expression of a novel, phosphoinositide-binding amphipathic helix, Kalrn promoter usage is expected to affect function.