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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.

Penta EF-hand (PEF) proteins apoptosis-linked gene 2 (ALG-2) and peflin are cytoplasmic Ca 2+ sensors for which physiological roles are still emerging. Here we demonstrate that adjustment of the ALG-2:peflin expression ratio can modulate basal ER export rates up or down by 107% of the basal rate. Through their ALG-2 subunit, ALG-2-peflin hetero-oligomers are shown to bind ERES and inhibit ER export of multiple cargo types, including collagen I, while ALG-2 by itself binds ERES to stimulate cargo sorting and ER export. During sustained Ca 2+ signaling, peflin and ALG-2 are demonstrated to sharply lower ER export rates by 40% in a novel secretory response to demanding physiological conditions. However, in highly Ca 2+stressed cells, peflin's suppressive role appears to promote pro-apoptotic unfolded protein response (UPR) signaling, indicating that the regulation is not necessarily pro-survival. Thus, regulation of secretion by PEF protein sub-complex binding to ERES in response to Ca 2+ signals impacts key cellular decision processes relevant to aging, neurodegeneration and diabetes.

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