title: An N-terminal Amphipathic Helix Binds Phosphoinositides and Enhances Kalirin Sec14 Domain-mediated Membrane Interactions.
abstract: Previous studies revealed an essential role for the lipid-binding Sec14 domain of kalirin (KalSec14), but its mechanism of action is not well understood. Because 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, whereas promoter C encodes an amphipathic helix (Kal-C-helix). Both bKalSec14 and cKalSec14 interacted with lipids in 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. Although cKalSec14 showed significantly reduced binding to liposomes lacking phosphatidylinositol phosphates or cholesterol, liposome binding by bKalSec14 and cKalSec14KKED was not affected. When expressed in AtT-20 cells, bKalSec14-GFP was diffusely localized, whereas cKalSec14-GFP localized to the trans-Golgi network 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.
authors: From the Departments of Neuroscience and.
authors: Megan B
authors: MB
authors: Miller
authors: From the Departments of Neuroscience and.
authors: Kurutihalli S
authors: KS
authors: Vishwanatha
authors: From the Departments of Neuroscience and.
authors: Richard E
authors: RE
authors: Mains
authors: From the Departments of Neuroscience and Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut 06030 eipper@uchc.edu.
authors: Betty A
authors: BA
authors: Eipper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D005455:Fluorescent Antibody Technique; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D010911:Pituitary Neoplasms / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D020662:Guanine Nucleotide Exchange Factors; C102365:Kalrn protein, rat; D008074:Lipoproteins; D008081:Liposomes; D010716:Phosphatidylinositols; C486622:Sec14l2 protein, rat; D015534:Trans-Activators
keywords: CRAL_TRIO; Golgi; PI(4)P; cholesterol; liposome; phosphoinositide; secretion; trans-Golgi network