Rab3gap1 palmitoylation cycling modulates cardiomyocyte exocytosis and atrial natriuretic peptide release

Rab3 GTPase-activating protein 1 (Rab3gap1) hydrolyzes GTP on Rab3 to inactivate it and reinitiate the Rab3 cycle, which regulates exocytic release of neuropeptides and hormones from neuroendocrine cells and atrial natriuretic peptide (ANP) secretion by cardiomyocytes. Cysteine palmitoylation of Rab3gap1 by the Golgi-localized S-acyltransferase zDHHC9 was recently shown to hinder ANP release by impairing Rab3gap1-mediated nucleotide cycling on Rab3a. Here, we interrogate the cysteine residues of Rab3gap1 modified by palmitoylation and impacts on ANP secretion in cardiomyocytes. Although mutation of the previously identified cysteine (Cys)-678 site of Rab3gap1 alone was insufficient to elicit complete loss of Rab3gap1 palmitoylation in cardiomyocytes, combinatorial mutation of Cys-509, 510, 521, 522, and 678 (Rab3gap1^5CS) dramatically reduced Rab3gap1 palmitoylation. Notably, total cellular GTPase-activating protein (GAP) activity in cardiomyocytes was maintained with mutation of the Rab3gap1 palmitoylation sites as the Rab3gap1^5CS mutant substantially reduced steady-state Rab3a-GTP levels in cardiomyocytes similar to wild-type Rab3gap1. However, although expression of wild-type Rab3gap1 induced robust secretion of ANP and greatly enhanced phenylephrine-stimulated ANP release, the Rab3gap1^5CS palmitoylation-deficient mutant was incapable of promoting exocytosis and ANP release by cardiomyocytes. These data suggest Rab3gap1 cysteine palmitoylation may target Rab3gap1 to Rab3a for regulated GAP-mediated inactivation at specific intracellular membrane domains to modulate the Rab3 cycle and exocytosis. Collectively, these data support a role for Rab3gap1 palmitoylation cycling in spatiotemporal control of the Rab3 cycle to regulate exocytosis and ANP secretion by cardiomyocytes.