Effect of ionomycin on interaction of calnexin with vesicular stomatitis virus glycoprotein is cell type-specific

Abstract

Ionomycin is a calcium ionophore that induces release of calcium ions (Ca IntroductionThe endoplasmic reticulum (ER) is the major intracellular reservoir of calcium ions. The total concentration of calcium in the ER is on the order of millimoles. In comparison, the calcium ion concentration in cytoplasm is three orders of magnitude lower (1). Approximately 50% of calcium ions in the ER can be rapidly released by the action of inositol 4,5-bisphosphate. The fraction of stored calcium that is not sensitive to inositol 4,5-bisphosphate is released into the cytoplasm by calcium ionophores. Drainage of calcium ions from the ER by calcium ionophores can cause rapid secretion of ER-resident proteins (2,3). A previous report found that the ER-resident protein immunoglobulin joining chain (J-chain) was released from ER-retention mechanisms and secreted outside cells by incubating J-chain-transfected fibroblasts with the calcium ionophore ionomycin (4).Vesicular stomatitis virus glycoprotein (VSV-G) is a type I membrane protein and has been used as a cargo molecule for research on ER-Golgi transport (5). VSV-G molecules reside in the ER until they are folded properly to form a homotrimer and exported outside the ER (6,7). Unfolded VSV-G molecules are retained in the ER and eventually degraded by the ubiquitin-proteasome system, a mechanism referred to as the ER quality control system (8,9). Chaperone molecules have a very important role in this system: they form a molecular complex with unfolded proteins in the ER lumen. When the cargo molecules are folded properly, this molecular complex is resolved and the cargo proteins are exported. The chaperone molecules responsible for VSV-G retention in the ER are immunoglobulin heavy chain-binding protein (BiP) and calnexin (10). Although both the interaction of chaperone with cargo molecule and the anterograde transport of VSV-G are regulated by calcium ions (11), the direct effects of calcium deprivation and ER-Golgi transport of VSV-G have not been carefully studied.In the present study, we investigated the effects of the calcium ionophore ionomycin on ER-Golgi transport of VSV-G. In both baby hamster kidney (BHK) and HeLa cells, ionomycin inhibited VSV-G transport. However, in contrast to findings for HeLa cells, interaction of VSV-G with calnexin was not responsible for delayed transport of VSV-G in BHK cells. These results indicate that calcium-dependent transport is regulated differently in BHK and HeLa cells. Materials and MethodsCells BHK cells (a hamster-derived fibroblastic cell line) and HeLa cells (a human-derived fibroblastic cell line) were cultured with 10% fetal calf serum-Dulbecco's modified Eagle's medium (10% FCS-DMEM) supplemented with 50 U/mL penicillin and 50 μg/mL streptomycin.

Chaperoning the Mononegavirales: Current Knowledge and Future Directions

“…As in the cytoplasm, inhibiting protein folding in the ER is likely to have broad antiviral activity against the Mononegavirales due to their general use of this compartment for the generation of essential glycoproteins. Indeed, non-specific interference with protein folding in the ER by altering calcium levels or by blocking key glycosylation enzymes that prevent folding have been reported to block the maturation of VSV [107,114], SV5 [100], MeV [97], and EBOV [115]. In addition, compounds that inhibit multiple chaperones, including those present in the ER, such as Sorafenib, OSU-0312, or epigallocatechin gallate, can reduce the replication of EBOV, MeV, and MuV infection [98,103,104,116].…”

Chaperoning the <em>Mononegavirales</em>: Current Knowledge and Future Directions

“…As in the cytoplasm, inhibiting protein folding in the ER is likely to have broad antiviral activity against the Mononegavirales due to their general use of this compartment for generation of essential glycoproteins. Indeed, non-specific interference with protein folding in the ER by altering calcium levels or by blocking key glycosylation enzymes that prevent folding have been reported to block the maturation of VSV [102,114], SV5 [115], MeV [91], and EBOV [116]. In addition, compounds that inhibit multiple chaperones, including those present in the ER, such as Sorafenib, OSU-0312, or (-)epigallocatechin gallate, can reduce the replication of EBOV, MeV, and MuV infection [92,93,117,118].…”

RANK Deficiency Ameliorates Podocyte Injury by Suppressing Calcium/Calcineurin/ NFATc1 Signaling

“…Ionomycin is a calcium ionophore which can increase intracellular calcium ions [23] and is widely used as a tool to induce podocyte injury [24]. To observe whether RANK was changed in response to podocyte injury in vitro, we studied its expression in ionomycintreated podocytes.…”