Abstract
SummaryEeyarestatin 1 (ES1) inhibits p97-dependent protein degradation, Sec61-dependent protein translocation into the endoplasmic reticulum (ER), and vesicular transport within the endomembrane system. Here, we show that ES1 impairs Ca2+ homeostasis by enhancing the Ca2+ leakage from mammalian ER. A comparison of various ES1 analogs suggested that the 5-nitrofuran (5-NF) ring of ES1 is crucial for this effect. Accordingly, the analog ES24, which conserves the 5-NF domain of ES1, selectively inhibited protein translocation into the ER, displayed the highest potency on ER Ca2+ leakage of ES1 analogs studied and induced Ca2+-dependent cell death. Using small interfering RNA-mediated knockdown of Sec61α, we identified Sec61 complexes as the targets that mediate the gain of Ca2+ leakage induced by ES1 and ES24. By interacting with the lateral gate of Sec61α, ES1 and ES24 likely capture Sec61 complexes in a Ca2+-permeable, open state, in which Sec61 complexes allow Ca2+ leakage but are translocation incompetent.
Highlights
The endoplasmic reticulum (ER) is the intracellular organelle in which most secretory and many transmembrane proteins are folded and assembled in eukaryotic cells (Cross et al, 2009b; Lang et al, 2017; Rapoport et al, 2017)
Since ER Ca2+ leakage can be unmasked by inhibiting sarco/ER Ca2+ ATPases (SERCA) in multiple cell types, it is generally assumed that [Ca2+]ER is maintained by balancing ER Ca2+ leakage and ATP-dependent Ca2+ influx into the ER via SERCA pumps (Camello et al, 2002; Carreras-Sureda et al, 2018)
Eeyarestatin 1 (ES1) Depletes ER Ca2+ by Enhancing Ca2+ Leakage from ER To obtain an integrated view of the Ca2+ status in cells under treatment with ES1, we measured free Ca2+ concentrations simultaneously in the ER ([Ca2+]ER) and the cytosol ([Ca2+]cyt) in the absence of extracellular Ca2+ to minimize Ca2+ entry. [Ca2+]ER was imaged with the genetically encoded, FRET-based Ca2+ sensor D1ER (Palmer et al, 2004), which was stably expressed in HEK cells (HEK-D1ER)
Summary
The endoplasmic reticulum (ER) is the intracellular organelle in which most secretory and many transmembrane proteins are folded and assembled in eukaryotic cells (Cross et al, 2009b; Lang et al, 2017; Rapoport et al, 2017). Studies revealed that this organelle stores Ca2+ in an ATP-dependent manner (Carreras-Sureda et al, 2018). The total Ca2+ stored in ER are in the order of 1–3 mM and the luminal free Ca2+ concentration in ER ([Ca2+]ER) can reach 100–800 mM, which represents more than 1,000-fold of the cytosolic Ca2+ concentration ([Ca2+]cyt) at rest (Carreras-Sureda et al, 2018). Since ER Ca2+ leakage can be unmasked by inhibiting sarco/ER Ca2+ ATPases (SERCA) in multiple cell types, it is generally assumed that [Ca2+]ER is maintained by balancing ER Ca2+ leakage and ATP-dependent Ca2+ influx into the ER via SERCA pumps (Camello et al, 2002; Carreras-Sureda et al, 2018). The molecular structure of Ca2+ leak channels in the ER has not yet been fully elucidated (Camello et al, 2002), but it is remarkable that the ubiquitously expressed Sec complex has been identified as a high-conductance Ca2+-permeable channel that can support Ca2+ leakage from the ER
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