Vesicular Calcium Regulates Coat Retention, Fusogenicity, and Size of Pre-Golgi Intermediates

Abstract

This study establishes a role for luminal Ca ²⁺ in ER/Golgi transport organelles and elucidates an effector mechanism involving the EF-hand protein ALG-2 and regulation of COPII coat retention.

Abstract

The significance and extent of Ca ²⁺ regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca ²⁺ with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca ²⁺ in the early secretory pathway. Specific depletion of luminal Ca ²⁺ in living normal rat kidney cells using cyclopiazonic acid (CPA) resulted in the extreme expansion of vesicular tubular cluster (VTC) elements. Consistent with this, a suppressive role for vesicle-associated Ca ²⁺ in COPII vesicle homotypic fusion was demonstrated in vitro using Ca ²⁺ chelators. The EF-hand–containing protein apoptosis-linked gene 2 (ALG-2), previously implicated in the stabilization of sec31 at endoplasmic reticulum exit sites, inhibited COPII vesicle fusion in a Ca ²⁺-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca ²⁺ on homotypic fusion. Immunoisolation established that Ca ²⁺ chelation inhibits and ALG-2 specifically favors residual retention of the COPII outer shell protein sec31 on pre-Golgi fusion intermediates. We conclude that vesicle-associated Ca ²⁺, acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca ²⁺-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.