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Abstract

Calreticulin (CRT) is an endoplasmic reticulum chaperone protein that is involved in quality control process during protein folding and maturation. It plays an important role as a regulator of intracellular calcium homeostasis. Previously it has been shown that loss of CRT protein results in endoplasmic reticulum stress, increase in ubiquitin-proteasome activity, and resistance to apoptosis. Our preliminary studies illustrated that CRT deficient cells expresses significantly higher connexin 43 protein, however its function is significant suppressed. We showed that connexin 43 was accumulated within intracellular vesicles. Because connexin 43 function is dependent on its trafficking and localization, we hypothesized that loss of CRT function increases rate of endocytosis via caveolin dependent pathway. To test our hypothesis we measured the rate of uptake of fluorescently tagged-Wheat Germ Agglutinin, in wild type (wt) versus crt-/- mouse embryonic fibroblast cells (MEF). In crt-/- cells WGA was completely endocytosed within 30 minute incubation while in wt cells WGA was localized both on cell membrane and intracellular vesicles at 30 minute. The endocytotic process in crt -/- was significantly reduced by inhibiting proteasome activity with MG132. The caveolin-1 mRNA and protein levels were significantly increased in crt-/- cells compared to wt cells. However, there were no significant changes in clathrin protein level in wt versus crt-/- cells. Using Methyl-B-cylcodextrin to deplete cell cholesterol content, we demonstrated inhibition of endocytosis in crt-/- cells which confirms a major role for caveolin in endocytosis in these cells. Overall our data are the first to illustrate the inhibitory role of CRT in endocytotic pathways. Acknowledgment: This research was funded by Qatar National Research Fund (NPRP4-043-3-016).

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/content/papers/10.5339/qfarc.2014.HBPP1034
2014-11-18
2020-01-26
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http://instance.metastore.ingenta.com/content/papers/10.5339/qfarc.2014.HBPP1034
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