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150-kDa oxygen-regulated protein (ORP150) functions as a novel molecular chaperone in MDCK cells
1Department of Anatomy and Neuroscience, Osaka University Graduate School of Medicine, Suita City, Japan. ybando@anat2.med.osaka-u.ac.jp
Abstract:
To assess the participation of the 150-kDa oxygen-regulated protein (ORP150) in protein transport, its function in Madin-Darby canine kidney (MDCK) cells was studied. Exposure of MDCK cells to hypoxia resulted in an increase of ORP150 antigen and increased binding of ORP150 to GP80/clusterin (80-kDa glycoprotein), a natural secretory protein in this cell line. In ORP150 antisense transformant MDCK cells, GP80 was retained within the endoplasmic reticulum after exposure to hypoxia. Metabolic labeling showed the delay of GP80 maturation in antisense transformants in hypoxia, whereas its matured form was detected in wild-type cells, indicating a role of ORP150 in protein transport, especially in hypoxia. The affinity chromatographic analysis of ORP150 suggested its ability to bind to ATP-agarose. Furthermore, the ATP hydrolysis analysis showed that ORP150 can release GP80 at a lower ATP concentration. These data indicate that ORP150 may function as a unique molecular chaperone in renal epithelial cells by facilitating protein transport/maturation in an environment where less ATP is accessible.
Insights
The 150-kDa oxygen-regulated protein (ORP150) acts as a molecular chaperone in kidney cells. It aids in protein transport and maturation, particularly under low ATP conditions during hypoxia.
Area of Science:
- Cell Biology
- Molecular Biology
- Renal Physiology
Background:
- The 150-kDa oxygen-regulated protein (ORP150) is involved in cellular stress responses.
- Protein transport and maturation are critical cellular processes, especially under hypoxic conditions.
Purpose of the Study:
- To investigate the role of ORP150 in protein transport within Madin-Darby canine kidney (MDCK) cells.
- To elucidate the mechanism of ORP150 function, particularly its interaction with secretory proteins.
Main Methods:
- Studied ORP150 expression and binding to GP80/clusterin in MDCK cells under hypoxia.
- Utilized ORP150 antisense transformant cells to assess GP80 retention and maturation.
- Employed metabolic labeling and affinity chromatography to analyze protein transport and ATP binding.
Main Results:
- Hypoxia increased ORP150 levels and its binding to GP80 in MDCK cells.
- GP80 maturation was impaired in ORP150-deficient cells under hypoxia, with retention in the endoplasmic reticulum.
- ORP150 demonstrated ATP-binding and hydrolysis capabilities, releasing GP80 at low ATP concentrations.
Conclusions:
- ORP150 functions as a molecular chaperone in renal epithelial cells.
- It facilitates protein transport and maturation, especially during hypoxia when ATP levels are reduced.
- ORP150 plays a crucial role in maintaining cellular function under stressful conditions.