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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02951

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Ubiquitin-proteasome-dependent degradation of mammalian ER stearoyl-CoA desaturase

Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan

View larger version (45K): [in a new window]   Fig. 1. Degradation of exogenously expressed SCD1 is inhibited by proteasome inhibitors. CHO-K1 cells or HeLa cells were transfected with plasmids harboring SCD1-HA or TCR-flag cDNAs. After 48 hours culture, the cells were pulsed with [35S]methionine in the presence of DMSO or 50 µM MG132 (A), and 20 or 100 µM leupeptin or 2 µM epoxomicin (epox) (B) for 30 minutes, then subjected to the chase reaction under the indicated conditions for the indicated times. Proteins (150 µg) were immunoprecipitated using anti-HA (for SCD1-HA) or anti-FLAG (for TCR-FLAG) antibodies and analyzed with an SDS-PAGE and BAS-2500 imager. Band intensities corresponding to SCD1-HA and TCR-flag were quantified by Image Gauge and shown by percentages of the signals setting those at time zero as 100%. The results shown are the average of three independent experiments. Asterisks: nonspecific immunoprecipitation bands.

View larger version (30K): [in a new window]   Fig. 2. Endogenously expressed SCD1 is also degraded by proteasomes. NIH 3T3-L1 cells were grown in differentiation cocktail containing methylisobutylxanthine, dexamethasone, and insulin. After 10 d, the cells were collected and subjected to (A) SDS-PAGE and immunoblot analysis using anti-SCD1 antibody, and (B) pulse-chase analysis using [35S]methionine in the absence (DMSO) or presence of 50 µM MG132, as described in Fig. 1 and Materials and Methods. Proteins were immunoprecipitated by anti-SCD1 antibody and analyzed by SDS-PAGE and subsequent digital autoradiography using BAS2500. Other conditions were the same as in Fig. 1.

View larger version (59K): [in a new window]   Fig. 3. Polyubiquitylated SCD1 accumulates in the presence of MG132. (A) CHO-K1 cells were transfected with pcDNA3.1 or the plasmid harboring SCD1-HA and grown for 48 hours. DMSO or 50 µM MG132 was then added to the cells and culture was continued for 5 hours. The cells were solubilized with buffer containing SDS (see Materials and Methods) and subjected to immunoprecipitation using anti-HA antibodies. The immunoprecipitates were analyzed by SDS-PAGE and subsequent immunoblotting with anti-polyubiquitin antibodies (upper panel) or anti-HA antibodies (lower panel). Lane 1 (IP control): immunoprecipitation without cell lysate. (B) NIH 3T3-L1 cells were grown in the differentiation cocktail for 10 days, DMSO or 50 µM MG132 was added, and culture continued for 5 hours. The cells were subjected to immunoprecipitation using anti-SCD1 C-terminal peptide antibodies or preimmune IgG and the immunoprecipitates were analyzed by SDS-PAGE and subsequent immunoblotting with anti-polyubiquitin (upper panels) or SCD1-C-peptide antibodies (lower panels). Lane 1 (IP control): immunoprecipitation without cell lysate. (C) CHO-K1 cells were transfected with the indicated plasmids and grown for 48 hours. MG132 (50 µM) was added to the cells and culture was continued for 5 hours. The cells were solubilized using SDS-buffer and subjected to immunoprecipitation using anti-Myc antibody. The immunoprecipitates were analyzed by SDS-PAGE and subsequent immunoblotting with anti-HA antibody. Magic Mark XP (Invitrogen) containing IgG-binding sequence-tagged proteins was used as molecular mass markers; detected by immunoblotting.

View larger version (39K): [in a new window]   Fig. 4. Polyubiquitylated SCD1 accumulates in the ER in the presence of MG132. (A) CHO-K1 cells were transfected with pcDNA3.1 or the plasmid harboring SCD1-HA cDNA, and grown for 48 hours. DMSO or 50 µM MG132 was then added to the cells and the culture continued for 5 hours. Post-nuclear supernatant (PNS) was prepared from the cells, and then fractionated into the supernatant (S) and membrane (P) fractions. Both fractions were subjected to immunoprecipitation using anti-HA antibody and the immunoprecipitates were then analyzed by SDS-PAGE and subsequent immunoblotting using anti-polyubiquitin antibody (upper panel). The filter was re-blotted with anti-HA antibody (middle panel), or anti-p97 antibody (lower panel). Lanes 1-4: 5% of PNS prepared from the cells grown under the indicated conditions were directly applied. Lane 13 (IP control): immunoprecipitation without cell lysate. (B) Quantification of p97 recovered to the ER of the SCD1-HA-expressing cells. Immunoprecipitation was performed as described in A, the gel was analyzed by digital autoradiography, and band intensities were calculated setting that of the total amount of p97 as 100%. The average of three independent experiments is shown.

View larger version (57K): [in a new window]   Fig. 5. The N-terminal PEST sequence-containing region of SCD1 is responsible for the proteasomal degradation. (A) The PEST sequence regions in the N-terminal segment of SCD1 predicted by the PEST-Find algorithm (shown in red characters). (B) CHO-K1 cells were transfected with plasmids carrying SCD1-HA cDNA or 66 SCD1-HA cDNA and grown for 48 hours. The cells were lysed with SDS buffer and analyzed by SDS-PAGE and subsequent immunoblotting with anti-HA antibody. (C) Indirect-immunofluorescence microscopy. Fluorescence images of SCD1-HA and 66 SCD1-HA (green) and calnexin (red) were taken using a confocal microscope. Merged images are also shown. Bar, 10 µm. (D) The cells obtained above were subjected to pulse-chase analysis with [35S]methionine in the presence of DMSO or 2 µM epoxomicin. At the indicated time points, proteins were immunoprecipitated using anti-HA antibody, resolved by SDS-PAGE, and analyzed using a BAS2500 Image Analyzer. Band intensities were quantified by Image Gauge and are shown as the percentage of the signal at time zero. The results shown are the average of three independent experiments.

View larger version (42K): [in a new window]   Fig. 6. The 66-residue N-terminal segment of SCD1 appended to a soluble reporter protein fails to stimulate degradation. (A) CHO-K1 cells were transfected with the plasmids harboring the indicated cDNAs, grown for 48 hours, and subjected to immunofluorescence microscopy. Fluorescence images of EGFP (green) and calnexin (red) were taken using a confocal microscope. Merged images are also shown. Bar, 10 µm. (B) The cells grown as above were subjected to pulse-chase analysis. Proteins were immunoprecipitated by anti-EGFP antibody. Other conditions were as described in Fig. 5D. (C) CHO-K1 cell were transfected with EGFP, SCD66-EGFP, SCD33-EGFP, or SCD1wt-EGFP constructs. After 48 hours, the transfected cells were treated with cycloheximide (10 µg/ml) for 0, 2, and 4 hours and their fluorescence was determined using flow cytometric analysis. The results shown are the average of three independent experiments.

View larger version (56K): [in a new window]   Fig. 7. The 66-residue N-terminal region of SCD1 makes an otherwise stable ER membrane protein unstable. (A) CHO-K1 cells were transfected with plasmids carrying b5-EGFP cDNA or SCD66-b5-EGFP cDNA, grown for 48 hours, and analyzed by immunofluorescence microscopy. Fluorescence images of b5-EGFP and SCD66-b5-EGFP (green) and calnexin (red) were taken using a confocal microscope. Merged images are also shown. Bar, 10 µm. (B) Cells, prepared as above, were subjected to pulse-chase analysis with [35S]methionine in the absence (DMSO) or presence of 2 µM epoxomicin. Other conditions were as described in Fig. 5.

View larger version (31K): [in a new window]   Fig. 8. SCD1 is naturally short-lived and is constitutively degraded irrespective of cellular lipid levels that regulate SCD1 gene expression. (A) HEK-293 cells (untransfected) were grown for 36 hours in standard culture medium containing untreated FBS (delipidated, -) or delipidated FBS (delipidated, +). Total RNAs were isolated from the cells and the mRNA amount for the indicated proteins was determined by real-time quantitative PCR. Levels of mRNAs are shown relative to the amounts of mRNA from the cells cultured in untreated FBS-containing medium. The GAPDH gene was used as a negative control. (B) HEK-293 cells were transfected with plasmid carrying SCD1-HA cDNA, grown for 48 hours, and subjected to pulse-chase analysis with [35S]methionine under the indicated conditions. At the indicated chase times, cells were solubilized and subjected to immunoprecipitation with anti-HA antibodies. The immunoprecipitates were analyzed by SDS-PAGE and subsequent digital autoradiography. (C) Band intensities in B were quantified. Relative radioactivity is shown with the levels at time zero defined as 100%. The results shown are the average of three independent experiments.

View larger version (39K): [in a new window]   Fig. 9. Plasminogen does not influence degradation of SCD1 in cultured cells. (A) Hamster liver, hamster liver microsomes, CHO-K1 cells expressing PLG-flag or the indicated cells were subjected to SDS-PAGE and subsequent immunoblot analyses with anti-PLG antibodies. (B) CHO-K1 cells were transfected with a PLG-flag construct and subjected to subcellular fractionation by differential centrifugation after 48 hours culture. The fraction containing unbroken cells and nuclei was removed by centrifugation at 1,000 g for 10 minutes. The supernatant was fractionated sequentially at 6,000 g for 10 minutes and then 100,000 g for 30 minutes to obtain P1 and P2, respectively. P1 is composed of mitochondria and heavy microsomes, whereas P2 is composed of microsomes. Fractionated samples were analyzed by SDS-PAGE, and subsequent immunoblotting using anti-FLAG, anti-calnexin antibody (ER marker), or anti-Tim23 antibody (mitochondrial marker). (C) CHO-K1 cells were transfected with the indicated amounts of the empty vector (pcDNA3.1), or those harboring SCD1-HA and PLG-flag cDNAs. After 48 hours, the transfected cells were analyzed by SDS-PAGE and subsequent immunoblotting using anti-FLAG antibody or anti-HA antiserum. (D) CHO-K1 cells were transfected with pcDNA3.1 harboring SCD1-HA cDNA and different amounts of PLG-flag cDNA (total amounts were adjusted to 5 µg with pcDNA3.1). After 48 hours, the cells were subjected to [35S]methionine pulse-chase analysis for the indicated time intervals. The results shown are the average of three independent experiments. Other conditions were as described in Fig. 5D. (E) The extracts obtained from CHO-K1 cells expressing PLG-flag or from untransfected HepG2 cells were resolved by SDS-PAGE and analyzed by immunoblotting using anti-PLG, or anti-calnexin antibodies. (F) The extracts obtained from control (RNAi for EGFP) or PLG-knockdown HepG2 cells were analyzed by SDS-PAGE and subsequent immunoblotting using anti-PLG- or anti-GAPDH antibodies. PLG was depleted by 93%. (G) Hep G2 cells were subjected to RNAi for PLG and pCMV SCD1-HA plasmid transfection as described in Materials and Methods. The cells were then subjected to [35S]methionine pulse-chase experiments as

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