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.
Highlights
Promoter usage determines the peptide preceding the lipid-binding KalSec14 domain
Kalirin C-promoter encodes an amphipathic helix, which interacts with phosphoinositides, localizes to the trans-Golgi network, alters KalSec14 interactions with cell membranes, and stimulates secretion
Alternative Promoter Usage Creates Two Structurally Distinct KalSec14 Domains—Full-length kalirin transcripts are initiated at one of four promoters, termed A, B, C, and D (Fig. 1A); transcripts generated from the different promoters encode 4 –38 amino acids that precede exon 2
Summary
Results: Kalirin C-promoter encodes an amphipathic helix, which interacts with phosphoinositides, localizes to the trans-Golgi network, alters KalSec interactions with cell membranes, and stimulates secretion. Conclusion: Sec domain function is altered by the preceding phosphoinositide-binding amphipathic helix. Promoter B encodes a charged, unstructured peptide, whereas promoter C encodes an amphipathic helix (Kal-Chelix) Both bKalSec and cKalSec interacted with lipids in PIP strip and liposome flotation assays, with significantly greater binding by cKalSec in both assays. Through its ability to control expression of a novel, phosphoinositide-binding amphipathic helix, Kalrn promoter usage is expected to affect function. Alternative promoter usage generates isoforms of kalirin in which its CRAL_TRIO domain is preceded by four very different sequences encoded by unique initial exons: Ex1A, Ex1B, Ex1C, or Ex1D (Fig. 1A). We show that these N-terminal peptides affect Sec domain lipid binding, subcellular localization, and function
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