Perturbation of cellular calcium blocks exit of secretory proteins from the rough endoplasmic reticulum
1Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
Abstract:
In the cultured human hepatoma HepG2, Ca2+ ionophores block secretion of different secretary proteins to different extents, alpha 1-antitrypsin secretion being more sensitive to A23187 and ionomycin than is alpha 1-antichymotrypsin, and albumin secretion the least of the three proteins studied. As judged by subcellular fractionation experiments and by treatment of pulse chase labeled protein with endoglycosidase H, A23187 and ionomycin cause newly made secretory proteins to remain within the rough endoplasmic reticulum (ER). Experiments in which A23187 is added at different times during a pulse or chase show that secretion of newly made alpha 1-antitrypsin becomes resistant to the ionophore, on average, 15 min after synthesis; this is about 20 min before it reaches the trans-Golgi, and while it is still within the rough ER. We speculate that a high concentration of Ca2+ within the ER may be essential for certain secretory proteins to fold properly, that folding is inhibited when ER Ca2+ levels are lowered by ionophore treatment, and that unfolded proteins, particularly alpha 1-antitrypsin, cannot exit the rough ER. Treatment of murine 3T3 fibroblasts or human hepatoma HepG2 cells with the Ca2+ ionophores A23187 or ionomycin also induces a severalfold accumulation of the ER lumenal protein Bip (Grp78). These findings disagree with a recent report that Ca2+ ionophores cause secretion of Bip and other resident ER proteins, but is consistent with other reports that A23187 causes accumulation of mRNAs for Bip and other ER lumenal proteins.
Insights
Calcium ionophores block protein secretion by retaining proteins in the endoplasmic reticulum (ER). This suggests that ER calcium is crucial for proper protein folding and secretion, impacting proteins like alpha-1-antitrypsin.
Area of Science:
- Cell Biology
- Biochemistry
Background:
- Secretory proteins are synthesized and processed through the endoplasmic reticulum (ER) and Golgi apparatus.
- Calcium ions (Ca2+) play critical roles in various cellular processes, including protein folding and trafficking.
- The precise role of ER calcium in protein secretion remains an area of investigation.
Purpose of the Study:
- To investigate the effect of calcium ionophores on the secretion of specific proteins from human hepatoma HepG2 cells.
- To determine the cellular location where calcium ionophores impede protein secretion.
- To explore the potential role of ER calcium concentration in protein folding and ER exit.
Main Methods:
- Cultured human hepatoma HepG2 cells were treated with calcium ionophores A23187 and ionomycin.
- Subcellular fractionation and endoglycosidase H treatment were used to analyze protein localization and modification.
- Pulse-chase labeling experiments tracked the synthesis and secretion of specific proteins over time.
- Accumulation of ER lumenal protein Bip (Grp78) was assessed in response to ionophore treatment.
Main Results:
- Calcium ionophores differentially blocked the secretion of alpha 1-antitrypsin, alpha 1-antichymotrypsin, and albumin, with alpha 1-antitrypsin being most sensitive.
- Ionophore treatment caused newly synthesized secretory proteins to accumulate within the rough ER.
- Secretion of alpha 1-antitrypsin became resistant to ionophore treatment approximately 15 minutes post-synthesis, while still within the rough ER.
- A significant accumulation of the ER lumenal protein Bip (Grp78) was observed.
Conclusions:
- Calcium ionophores disrupt the secretory pathway by retaining proteins in the rough ER.
- Lowered ER calcium levels likely inhibit proper protein folding, preventing ER exit.
- These findings highlight the importance of ER calcium for efficient protein folding and secretion, particularly for proteins like alpha 1-antitrypsin.
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