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Increased rate of endocytosis in calreticulin knockout MEF cells is mediated via Caevolin‐1 overexpression (LB195)
Author(s) -
Viswanathan Divya,
Pillai Dhanya,
Massaeli Hamid,
Mesaeli Nasrin
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb195
Subject(s) - endocytosis , calreticulin , microbiology and biotechnology , mg132 , endoplasmic reticulum , ubiquitin , proteasome , clathrin , intracellular , biology , endosome , chemistry , cell , proteasome inhibitor , biochemistry , gene
Calreticulin (CRT) is a highly expressed protein in mammalian cells, with both calcium binding and chaperone activity. As an endoplasmic reticulum chaperone, CRT is involved in quality control process during the folding and maturation of proteins. Previous reports illustrated that loss of CRT function (crt‐/‐) results in onset of ER stress, elevated ubiquitin‐proteasome activity, and increased resistance to apoptosis. We recently observed a significant increase in connexin 43 expression accompanied with a significant decrease in its function and accumulation of connexin 43 in intracellular vesicles. Thus we hypothesized that loss of CRT function increases rate of endocytosis. To test this hypothesis we examined rate of endocytosis using Wheat Germ Agglutining (WGA). Our data showed that in crt‐/‐ WGA was completely endocytosed within 30 minute incubation while in wild‐type cells WGA was localized both on cell membrane and intracellular vesicles at the same time. Furthermore, inhibiting proteasome with MG132 reduced endocytosis rate in crt‐/‐ cells. Western blot analysis illustrated a significant increase in caveloin‐1 protein level in crt‐/‐ cells. Quantitative real time PCR showed 3 fold increase in expression of caveolin‐1 in crt‐/‐ compared to wt cells. Furthermore, ubiquitin pull down experiment showed significantly higher caveolin‐1 ubiquitination in crt‐/‐ compared to wt cells. Our data show for the first time involvement of CRT in endocytotic pathways. Furthermore, we showed that knock out of CRT gene results in enhanced endocytosis that is dependent on the ubiquitin‐proteasome activity. Grant Funding Source : Qatar National Research Foundation