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

Abstract

This study establishes a role for luminal Ca2+ in ER/Golgi transport organelles and elucidates an effector mechanism involving the EF-hand protein ALG-2 and regulation of COPII coat retention.

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

“…5A, bar 10), which reportedly exhibits Sec31 binding but fails to bind several other ALG-2 targets (48), implies that a distinct ALG-2 target may be involved, as predicted by current models of PEF protein function, wherein an ALG-2 dimer cross-links and stabilizes the otherwise weak association of two effectors (47). This scenario is also consistent with the in vitro activity of ALG-2 to kinetically stabilize Sec31A at or near ERES and on COPII vesicles (10,32,33,35), which implies that ALG-2 cross-links Sec31A to an "anchor" site.…”

“…On the other hand, the distribution of Sec16A, a COPII scaffold that does not enter vesicles (46), was not affected by luminal Ca 2ϩ depletion (10). These findings were consistent with the enlarged spots representing aberrantly large early VTCs or abortive vesicle buds.…”

“…Depletion of Luminal Calcium-Conditions were identical to our extensively characterized protocol (10). Briefly, Ca 2ϩ -free DMEM (U.S.…”

“…Hence, Ca 2ϩ does not play a universal or necessarily mechanistically consistent role; secretion is a mosaic of Ca 2ϩ -dependent and -independent transport steps (23). At the ERto-Golgi stage, specific depletion of luminal Ca 2ϩ using the reversible SERCA inhibitor cyclopiazonic acid (CPA) caused Rbet1, a rapidly recycling ER/Golgi SNARE, to accumulate in large peripheral punctate structures (10). Despite this morphological perturbation, potential functional effects of luminal Ca 2ϩ depletion on anterograde ER/Golgi cargo transport remain to be established.…”

“…The PEF (penta-EF-hand-containing) protein apoptosis-linked gene-2 (ALG-2) acts as a Ca 2ϩ sensor at ER exit sites and, only when Ca 2ϩ is present, stabilizes association of Sec31 with the membrane (32)(33)(34)(35). We previously demonstrated that ALG-2 stabilizes residual Sec31 on COPII fusion intermediates in a Ca 2ϩ -dependent manner and that excess ALG-2 inhibited COPII vesicle fusion in a luminal Ca 2ϩ -dependent manner (10). Recently, an in vitro budding reconstitution using mammalian components demonstrated that excess ALG-2 attenuated budding in a Ca 2ϩ -dependent manner and that ALG-2 binding to Sec31A directly promoted Sec31A/ Sec23 interactions (36).…”