MUNC13 REGULATES SECRETION AT THE GOLGI IN A LOCALIZATION-DEPENDENT MANNER

Abstract

Background: Constitutive secretion is critical for the maintenance of eukaryotic cell structure and function. Our lab has shown that Rab34 is required for secretion at the Golgi^1, and that the C1 domain-containing protein, Munc13, is an effector of Rab34^2. Current studies seek to elucidate potential roles for Munc13in secretion at the Golgi.

Methods: Using a temperature-sensitive mutant of the Vesicular Stomatitis G-protein fused to GFP (VSVG-GFP) to monitor secretion, we examinedthe role of Munc13 in secretion in HeLa cells. Cells transfected with VSVG-GFP were treated with Munc13, amutant lacking the C1 domain (C1-less), and the phorbol esters TPA andPDBu. The rate of VSVG-GFP secretion was monitored using surface labelling of plasmalemmal VSVG-GFP and spinning disc confocal microscopy.

Results: TPA treatment resulted in an increase in the rate of VSVG-GFP appearance at the plasma membrane. Co-transfection of either Munc13 or C1-less alone also resulted in an increased rate of VSVG-GFP transport. Transfection of Munc13 plus TPA treatment resulted in amarked decrease in the rate of VSVG-GFP transport. Since TPA treatment relocalizes Munc13 to the plasma membrane, this result suggests that the availability of Munc13 in the cytosol is required for its effect on VSVG-GFP secretion.

Conclusions: Munc13 over-expression increases the rate of VSVG-GFP secretion to the plasma membrane. Sequestration of Munc13 at the plasma membrane with TPA abrogates thiseffect, and reduces the rate of VSVG-GFP secretion. We propose that Munc13 effects VSVG-GFP secretion via its interaction with Rab34 at the Golgi.

References:

1. Goldenberg, NM, S. Grinstein, M. Silverman. Golgi-bound Rab34 is a Novel Member ofthe Secretory Pathway. Mol BiolCell. 18(12):4762-4771 (2007).

2. Speight, P, M. Silverman.Diacylglycerol-Activated Hmunc13 Serves as an Effector of the GTPaseRab34. Traffic.6(10):858-865 (2005).