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. 2019 Apr 15;9:6066. doi: 10.1038/s41598-019-42331-6

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Clathrin- and polyubiquitin-independent peripheral quality control of PAS-mutants. (a) PAS-mutants are internalized by a clathrin-independent pathway. Internalization of hERG was measured by cell-surface ELISA in transiently transfected COS-7 cells. Clathrin-dependent internalization was inhibited by incubation in hypertonic media supplemented with 300 mM sucrose (15 min at 37 °C, followed by 45 min at 4 °C). Clathrin-dependent rapid internalization of transferrin receptor (TfR) used as positive control. (b) Effect of CHIP ablation on WT and PAS mutants steady-state expression 72 h post-transfection in HeLa cells. CHIP knockdown exceeded 95%. NT: non-target siRNA. Representative immunoblots shown (uncropped images in Supplementary Fig. S12). Solid line: different parts of the same gel. White space: separate gels. Mature complex-glycosylated and core-glycosylated (~135 kDa) hERGs indicated by solid and empty arrows, respectively. (c) The CHIP ubiquitin E3 ligase does not influence to the PAS-mutant cell-surface expression. hERG cell-surface density measured by PM-ELISA 48–72 h after transfection with non-target (siNT) or CHIP-specific (siCHIP) siRNA in HeLa cells. (d,e) CHIP is involved in peripheral quality control of drug-destabilized WT (d) but not PAS-mutant hERGs (e). Cell-surface turnover of untreated PAS-mutant hERG or WT-hERG unfolded by ouabain-induced intracellular K⁺-depletion (ouab, 300 nM) or by direct desipramine binding (des, 20 µM), monitored by PM-ELISA 48–72 h after transfection with non-target (siNT) or CHIP-specific (siCHIP) siRNA. (f) Poly-ubiquitination at the cell-surface is required for internalization of drug-destabilized WT but not PAS-mutant hERG. WT and F29L or T65 hERG were transiently co-expressed with excess wild-type ubiquitin (Ub) or a dominant-negative variant unable to form linked chains (Ub-DN). WT-hERG was unfolded by 2 h pre-treatment with ouabain (Ouab, 300 nM) or desipramine (Des, 10 µM). Overexpression of Ub-DN prevented rapid internalization of the drug-destabilized WT-hERG but had no effect on either PAS mutant. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See Methods for explanation of statistical analysis).