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First published online 15 August 2006
doi: 10.1242/jcs.03101

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Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle

¹ Laboratory of Molecular Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
² Laboratory of Clinical Pathobiology, Department of Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
³ Laboratory of Animal Genetics and Breeding, Department of Animal Science, Tokyo University of Agriculture, Atsugi 243-0034, Japan

View larger version (62K): [in a new window]   Fig. 1. Expression of wild-type and R664X AE1 in K562 cells. (A) Expression of bovine AE1 was examined by immunofluorescence with the monoclonal antibody cdb3-64 in K562bebWT, K562bebRX and control (K562C) cells. Bars, 10 µm. (B) K562bebWT and K562bebRX cells were pulse-labeled with [35S]methionine for 45 minutes and chased for the indicated periods. After cell-surface biotinylation, AE1 proteins were immunoprecipitated using tmb3-29 with (Surface) or without (Total) isolation on NutrAvidin beads. Migration of protein standards is indicated in kDa. Intensities of the bands at the indicated times for wild-type AE1 (WT, 105 kDa, and ) and R664X AE1 (R664X, 75 kDa, and ) in total ( and ) and cell-surface ( and ) fractions were determined by densitometric scanning and plotted (right). Data are the mean values of duplicate samples at each time point except for t=0 (n=3, mean ± s.d.). This procedure always produced a non-specific 77-kDa band (*) even in immunoprecipitates from control cells.

View larger version (38K): [in a new window]   Fig. 2. Dominant-negative effect of R664X AE1 on cell surface expression of wild-type AE1. (A) Wild-type () proteins in K562bebWT cells and wild-type () and R664X () proteins in K562bebWT/RX cells were pulse-labeled and chased for the indicated periods as in Fig. 1. The left figure shows immunoprecipitates from each sample after labeling without chase. The asterisk indicates a non-specific band. Data are the mean of duplicate samples at each time point. (B) K562bebWT, K562bebRX, K562bebWT/RX and K562C cells were transfected with bovine GPC and stained for AE1 and GPC with cdb3-64 and the anti-GPC antibody, respectively. Bars, 5 µm. (C) AE1 proteins in K562bebWT (WT), K562bebRX (RX), and K562bebWT/RX (WT/RX) cells were immunoprecipitated with monoclonal antibodies cdb3-64 or tmb3-26 (upper panels). Lane WT + RX contains immunoprecipitates from K562bebWT and K562bebRX cells mixed prior to solubilization for immunoprecipitation. The asterisk indicates what probably is an 70-kDa degradation product of wild-type AE1. Likewise, AE1 proteins synthesized in a cell-free translation system in the presence of canine pancreatic microsomes (Synthesized) were immunoprecipitated with tmb3-26 (lower right panel). Reactions contained wild type alone (WT), R664X alone (R664X), or both wild type and R664X AE1 (WT/RX). WT and RX reactions were combined prior to solubilization (WT + RX).

View larger version (29K): [in a new window]   Fig. 3. Dominant-negative effect of R664X AE1 on functional expression of wild-type AE1 in Xenopus oocytes. (A) 36Cl--uptake of oocytes injected with increasing amount of wild-type () or R664X () AE1 RNA. (B) 36Cl--uptake into oocytes injected with wild-type AE1 RNA (WT) with or without various amounts of R664X RNA (R664X). Values indicate DIDS-sensitive 36Cl--uptake calculated by subtracting the mean values in the presence of 10 µM DIDS (n=5-10) from values obtained in the absence of DIDS. Data are expressed as mean ± s.d. (n10). *P<0.05, **P<0.01 in oocytes injected with 0.5 ng or 1 ng/oocyte of wild-type RNA alone.

View larger version (34K): [in a new window]   Fig. 4. Increased stability of R664X AE1 in K562 cells in the presence of proteasome inhibitors. (A) K562bebWT and K562bebRX cells were incubated for 8 hours in the absence (None) or presence of the reagents indicated, and then wild-type and R664X protein levels were analyzed by immunoblotting with the tmb3-29 antibody. (B) K562bebWT and K562bebRX cells were pulse-labeled and chased for the indicated periods followed by immunoprecipitation of wild-type (WT) or R664X (R664X) AE1 in the presence (Lactacystin, ) or absence (None, ) of 10 µM lactacystin, as described in the legend for Fig. 1. (C) K562bebWT (WT) and K562bebRX (RX) cells were incubated for 8 hours in the absence (-) or presence (+) of 10 µM lactacystin and lysed in IP-buffer, followed by immunoprecipitation with cdb3-64. AE1 proteins and ubiquitylated proteins in detergent-soluble whole cell lysates (Whole, upper panels) or immunoprecipitates (IP, lower panels) were detected with the anti-AE1 (left) and anti-ubiquitin (right) antibodies, respectively. The detergent-insoluble fraction contained various ubiquitylated polypeptides but not proteins recognized with the AE1 antibody (data not shown). Blots were incubated with tmb3-29 (Whole) or anti-38K (IP) antibodies. Asterisks indicate non-specific signals observed in lysates from mock-treated cells (not shown). Migrating positions of the protein standards are shown in kDa.

View larger version (69K): [in a new window]   Fig. 5. Intracellular localization of wild-type and R664X AE1 in HEK293 cells. HEK293 cells were transiently co-transfected with the EGFP-wild-type (EGFP-WT) or EGFP-R664X (EGFP-R664X) AE1 and GPC (EGFP-WT/GPC or EGFP-RX/GPC) and stained for GPC with the anti-GPC antibody. HEK293 cells expressing EGFP-wild-type or EGFP-R664X AE1 (EGFP-WT or EGFP-RX) were also incubated with anti-calnexin (Calnexin), anti-GM130 (GM130), and anti-Lamp2 (Lamp2) followed by detection with a secondary antibody labeled with Alexa-Fluor-568. Bars, 10 µm.

View larger version (38K): [in a new window]   Fig. 6. Turnover of wild-type and R664X AE1 proteins in HEK293 cells. (A) Cell-surface proteins in HEK293 cells transiently transfected with wild-type (WT) and R664X (RX) AE1 were biotinylated and polypeptides in total cell lysate (T) were separated into bound (B) and unbound (U) fractions on NutrAvidin beads, followed by detection of AE1 proteins by immunoblotting. Each lane contained proteins from the equivalent volume of cell lysate. (B) HEK293 cells transiently transfected with wild type (WT), R664X (RX), wild type and GPC (WT/GPC), or wild-type and R664X AE1 (WT/RX) were pulse-labeled for 20 minutes and chased for the indicated periods followed by immunoprecipitation of AE1 as described for Fig. 1. Lanes 10+ contained immunoprecipitates from the cells chased for 10 hours in the presence of 10 µM lactacystin. The lower panel shows wild-type and R664X proteins remaining after the chase in the presence () or absence () of lactacystin. Data are the mean values of duplicate samples. Pulse-labeled wild-type and R664X AE1 in WT/GPC and WT/RX HEK293 cells showed stability similar to that observed in HEK293 cells transfected with wild type or R664X alone (data not shown).

View larger version (53K): [in a new window]   Fig. 7. The absence of ubiquitylated AE1 in transfected HEK293 cells. (A,B) HEK293 cells transiently transfected with wild-type (WT) and R664X (RX) AE1, their EGFP-tagged forms (EGFP-WT and EGFP-RX), EGFP-F508-CFTR (EGFP-F508), or empty vectors (Mock) were incubated for 8 hours in the absence (-) or presence (+) of 10 µM lactacystin. AE1 and EGFP-F508-CFTR were immunoprecipitated with cdb3-64 and the anti-CFTR, respectively. AE1 and EGFP-F508-CFTR in whole-cell lysates (A) and immunoprecipitates (B) were detected by immunoblotting with cdb3-64 and anti-CFTR (A) or anti-38K and anti-GFP (B, left panel), respectively. Ubiquitylated proteins in the immunoprecipitates were also reacted with the anti-ubiquitin (anti-Ub) antibody (B, right panel). Signals corresponding to high-molecular-weight species (HMW) were seen only on blots of EGFP-F508-CFTR. The asterisk indicates a non-specific product.

View larger version (92K): [in a new window]   Fig. 8. Distinct intracellular localization of EGFP-R664X AE1 and EGFP-F508-CFTR in HEK293 cells treated with lactacystin. Cells were transiently transfected with EGFP-R664X AE1 (EGFP-RX) or EGFP-F508-CFTR (EGFP-F508) and incubated with (B,C) or without (A) 10 µM lactacystin for 8 hours, and the intracellular localization of EGFP-tagged proteins was compared with that of calnexin (A,B) and ubiquitin (C). Bars, 10 µm.