John Sargeant,1 Danette Kowal Seiler,1 Tucker Costain,1 Corina T. Madreiter-Sokolowski,2 David E. Gordon,3 Andrew A. Peden,4 Roland Malli,2 Wolfgang F. Graier,2 and Jesse C. Hay1,∗


"1Division of Biological Sciences, Center for Structural and Functional Neuroscience, University of Montana, Missoula, Montana, USA
2Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria
3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA
4Department of Biomedical Science and Centre for Membrane Interactions and Dynamics, The University of Sheffield, Sheffield, United Kingdom
Jesse C. Hay: [email protected]
∗For correspondence: Jesse C. Hay [email protected]"


Calcium Imaging, Cell Biology


"ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed nonstop independent of Ca2+ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ 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 Ca2+, 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 Ca2+ 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 Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca2+ 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.

Keywords: calcium, vesicle trafficking, ER-to-Golgi transport, ALG-2, peflin, penta-EF-hand, calcium signaling, ER exit site, COPII, collagen
Abbreviations: ALG-2, apoptosis-linked gene-2; BHQ, 2,5-di-(t-butyl)-1,4-hydroquinone; CADEE, Ca2+-activated depression of ER export; ERES, ER exit site; NRK, normal rat kidney; PAEC, porcine aortic endothelial cell; PRR, proline-rich region; ROI, region of interest; SERCA, sarco-endoplasmic reticulum Ca2+ ATPase; TCF, total cell fluorescence; UPR, unfolded protein response

doi: DOI: 10.1016/j.jbc.2021.101393


"Image acquisition was completed by a Nikon TE300 inverted microscope equipped with a Nikon Plan Fluor 20×/0.75 objective, motorized high-speed Sutter Lambda filter wheel for emissions, CoolLED pe340 excitation system, and PCO Panda sCMOS camera, all automated with Micro-Manager software.

Nikon E800 microscope with an LED illumination unit (CoolLED pE 300white)"

Product Associated Features

The 340 nm and 380 nm LED illumination system provides the optimum excitation wavelengths for Fura-2-based calcium imaging, allowing high-precision, stable, high-throughput imaging with video-rate time resolution.

Product Type

pE-300white, pE-340fura


The Journal of Biological Chemistry

Year of Publication


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