ALG-2 Attenuates COPII Budding In Vitro and Stabilizes the Sec23/Sec31A Complex

Jonas M La Cour,Martin W Berchtold,Adam J Schindler,Randy Schekman,Makoto Kanzaki

doi:10.1371/journal.pone.0075309

Jonas M La Cour, Martin W Berchtold + Show 3 more

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https://doi.org/10.1371/journal.pone.0075309

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Abstract

Coated vesicles mediate the traffic of secretory and membrane cargo proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The coat protein complex (COPII) involved in vesicle budding is constituted by a GTPase, Sar1, the inner coat components of Sec23/Sec24 and the components of the outer coat Sec13/Sec31A. The Ca2+-binding protein ALG-2 was recently identified as a Sec31A binding partner and a possible link to Ca2+ regulation of COPII vesicle budding. Here we show that ALG-2/Ca2+ is capable of attenuating vesicle budding in vitro through interaction with an ALG-2 binding domain in the proline rich region of Sec31A. Binding of ALG-2 to Sec31A and inhibition of COPII vesicle budding is furthermore dependent on an intact Ca2+-binding site at EF-hand 1 of ALG-2. ALG-2 increased recruitment of COPII proteins Sec23/24 and Sec13/31A to artificial liposomes and was capable of mediating binding of Sec13/31A to Sec23. These results introduce a regulatory role for ALG-2/Ca2+ in COPII tethering and vesicle budding.

Highlights

Ca2+ is a ubiquitous messenger molecule regulating a wide array of cell biological processes
The significance of this interaction for endoplasmic reticulum (ER) to Golgi protein transport is not seen in the trafficking of VSV-G in cells depleted of ALG-2 [3]
In order to test the effect of ALG-2 on complex II (COPII) budding under controlled Ca2+conditions, we investigated the consequences of adding recombinant ALG-2 to a cell-free vesicle budding reaction in the presence or absence of free Ca2+

Summary

Introduction

Ca2+ is a ubiquitous messenger molecule regulating a wide array of cell biological processes. Recent evidence suggests that Ca2+ transients may be involved in regulatory mechanisms related to protein trafficking. The Ca2+ binding protein ALG-2 (product of the apoptosis linked gene-2) was found to interact with Sec31A, a component of the coat protein complex II (COPII) in a Ca2+dependent manner. It has been speculated that ALG-2 mediates a Ca2+-link to COPII dependent protein traffic [1,2,3]. Recent work by Bentley et al suggests that Ca2+ together with ALG-2 plays a regulatory role in the fusion of COPII vesicles after budding [5]. ALG-2 was originally assigned a functional role in apoptosis linking Ca2+ signaling to programmed cell death [6], no molecular mechanism underlying this function has been described. The suggested apoptotic function of ALG-2 was not confirmed by gene knock out studies in mice [7]

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