ALG-2 and peflin regulate COPII targeting and secretion in response to calcium signaling

Abstract

ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed nonstop independent of Ca 2+ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca 2+ signaling by adopting one of several distinct activity states. Functionally, these states can adjust the rate of ER export of COPII-sorted cargos up or down by ∼50%. We found that at steady-state Ca 2+ , ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. Upon Ca 2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration—phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca 2+ -mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca 2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca 2+ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.

Ca2+ regulation of constitutive vesicle trafficking

“…The reason for the opposite effect of peflin depletion in Sargeant et al . 39 and McGourty et al . 40 is not known.…”

“…Other cellular studies showed that recombinant ALG-2 bound to Sec31 was able to stabilize ERES 31 , 36 , and dominant-negative ALG-2 37 and depletion 38 studies also indicated a positive regulatory role. Recently, our lab helped reconcile some of these apparent contradictions by demonstrating that ALG-2 can either stimulate or inhibit ER-to-Golgi transport in the same system 39 . This duality in the regulatory role of ALG-2 may come down to multiple partners it can bind under different circumstances.…”

“…In the basal state, in the absence of significant cytosolic Ca 2+ oscillations, ALG-2 binds to the outer COPII coat subunit Sec31 31 , 32 , where it colocalizes with another PEF protein: peflin 39 , 40 . Likely, the colocalized peflin and ALG-2 at ERES are comprised of the earlier reported 1:1 heterodimer, observed in vitro to dissociate in response to Ca 2+ 41 .…”

“…Likely, the colocalized peflin and ALG-2 at ERES are comprised of the earlier reported 1:1 heterodimer, observed in vitro to dissociate in response to Ca 2+ 41 . Interestingly, depletion of peflin in NRK cells produced increased ALG-2 at ERES 38 and an increase in ER-to-Golgi trafficking of multiple COPII cargoes 39 , a situation reminiscent of ALG-2-binding to ERES in response to intracellular Ca 2+ oscillations 33 , indicating a straightforward positive and negative role for ALG-2 and peflin at ERES, respectively. Furthermore, double KD of ALG-2 and peflin did not decrease transport below control levels, indicating that neither is actually required for secretion and both are purely regulatory in this context.…”

“…Recently, our lab showed that distinct calcium signals and patterns can cause opposite effects on ER export in an ALG-2-dependent manner 39 . In NRK cells, exposure to histamine or ATP for up to 2.5 hours caused an ALG-2-dependent depression of ER-to-Golgi trafficking, but in neuroendocrine PC12 cells, the same treatment caused ALG-2-dependent enhancement of transport.…”