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First published online 3 January 2006
doi: 10.1242/jcs.02732

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Uncoupling proteasome peptidase and ATPase activities results in cytosolic release of an ER polytopic protein

Jon Oberdorf^*,, Eric J. Carlson^* and William R. Skach

Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA

View larger version (23K): [in a new window]   Fig. 1. Cytosolic release of TCA-insoluble CFTR fragments. CFTR degradation was carried out in the absence of inhibitors (A), or in the presence of 100 µM MG132 (B), 100 µM clasto-lactacystin ß-lactone, 500 µM GPFL and 100 µM leupeptin (C), 100 µM clasto-lactacystin ß-lactone (D), 100 µM ALLN (E), 40 µM hemin (F) or hexokinase and 2-deoxyglucose (G) as described in Materials and Methods. Total () and TCA-soluble () CFTR radioactive fragments recovered in the cytosolic fraction are expressed as the percentage of total CFTR counts present at t=0. The difference between total and TCA-soluble counts released (double arrow) indicates relative amount of CFTR released in the form of TCA-insoluble fragments. The gray line (panels B-G) shows total CFTR converted into TCA-soluble counts under control conditions.

View larger version (25K): [in a new window]   Fig. 2. Release of TCA-insoluble fragments is proportional to ß-subunit inhibition. (A-E) CFTR degradation was carried out at the indicated concentrations of MG132. The amount of total () and TCA-soluble () fragments released from ER membrane was determined as in Fig. 1. TCA-insoluble fragments released are indicated by a double arrow.

View larger version (71K): [in a new window]   Fig. 3. Released CFTR fragments are heterogeneous. (A) Supernatants of CFTR-degradation reactions were collected at the times indicated and analyzed by SDS-PAGE (7-12% gel) and autoradiography. Inhibitor concentrations were 100 µM for MG132, ALLN, clasto-lactacystin ß-lactone (ß lactone) and 40 µM for hemin. Hexokinase and 2-deoxyglucose were added to deplete ATP (–ATP). (B) Total products of the degradation reaction are shown to demonstrate the rate of disappearance of CFTR protein. High Mr material in lanes 26-30 (hemin treatment) represents ubiquitylated CFTR products (Xiong et al., 1999). Exposure of the top panels was longer (5x) than the bottom panels.

View larger version (25K): [in a new window]   Fig. 4. CFTR degradation involves simultaneous loss of multiple epitopes. The schematic shows the topological structure of CFTR and relative locations of N-terminal (N), C-terminal (C) and regulatory (R) domain epitopes. NDB, nucleotide-binding domain; *, location of methionine residues; open circles, N-linked glycosylation sites. (A) Control CFTR degradation assay performed as in Fig. 1. (B-D) Degradation products from panel A were immunoprecipitated with peptide-specific antisera raised against the N-terminus (), C-terminus () or R-domain () epitopes. Fragments recovered with each antibody were quantified by scintillation counting and expressed as the percentage of counts recovered at t=0.

View larger version (13K): [in a new window]   Fig. 5. Cytosolic fragments are derived from multiple peptide domains. Membrane pellet and supernatant fractions were collected from CFTR degradation reactions carried out for 2 hours in the presence of 100 µM MG132. Samples were immunoprecipitated by N-terminal, C-terminal and R-domain specific antisera and quantified by scintillation counting as in Fig. 5. Data are expressed as the percentage of total radioactive counts in pellet and supernatant fractions that remained associated with indicated epitopes.

View larger version (28K): [in a new window]   Fig. 6. Cytosolic CFTR fragments remain stably associated with proteasomes. (A) Supernatants from degradation reactions were collected after 2 hours in the presence of 100 µM MG132 and separated by glycerol gradient centrifugation. Fractions were collected top to bottom (1-14) and TCA-soluble () and TCA-insoluble () material was quantified by scintillation counting. (B) Samples from gradients in panel A were analyzed directly by SDS-PAGE (12-17% gel) and autoradiography. Migration of molecular mass markers in the gradient are indicated at top of the gel. (C) Degradation was carried out as in A and membranes were pelleted through a 0.5 M sucrose cushion. Supernatant and pellet fractions were analyzed directly (lanes 1,4) or immunoprecipitated with preimmune sera (PIS) or antisera against the 3 proteasome subunit.

View larger version (21K): [in a new window]   Fig. 7. p97 is inhibited by hemin and contributes to CFTR degradation. (A) Coomassie Blue-stained SDS gels of His-tagged wild type (WT) and dominant-negative mutant (QQ) p97 purified on a Ni-NTA column and run on glycerol gradients. (B) ATPase activity of purified wild-type p97, mutant p97 and 26S RRL proteasomes was determined in the presence and absence of 40 µM hemin as described in Materials and Methods. (C) CFTR degradation was carried out in the presence of indicated molar excess of dominant-negative p97 (QQ). Graph shows the fraction (mean ± s.e.m.) of newly synthesized CFTR that was converted into TCA-soluble peptide fragments.

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