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First published online 30 January 2007
doi: 10.1242/jcs.03351

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Characterization of the proteasome interaction with the Sec61 channel in the endoplasmic reticulum

¹ University of Cambridge, Cambridge Institute for Medical Research and Department of Clinical Biochemistry, Hills Road, Cambridge, CB2 2XY, UK
² Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK

View larger version (51K): [in this window] [in a new window]   Fig. 1. Proteasomes and ribosomes bind to different sites in the Sec61 channel. (A) JDY683 (pGAL-SEC61) derivatives with pCEN-LEU2-SEC61, or the same plasmid containing the indicated sec61 mutants, or the empty vector, were transformed with a reporter plasmid for co-translational import, pRS313-PHO8-URA3, or the reporter plasmid for post-translational import, pRS313-CPY-URA3, or the empty vector. Growth was monitored on SD containing glucose and uracil, but lacking leucine and histidine (right panels; this controls for Sec61p function) and the same plates without uracil (left panels). Growth in the absence of uracil indicates a translocation defect for the URA3 reporter. (B) Topology model of Sec61p. Position of mutations in mutant alleles used in the proteasome and ribosome binding experiments in C-E is indicated. (C) Yeast 19S RP (2 pmol) FLAG-tagged on Rpn11p were incubated with 20 eq of proteoliposomes from puromycin/high salt-treated yeast microsomes as described in Materials and Methods. 1 eq equals 1 µl of microsomes of A280=50. The membranes were floated through 1.8 M sucrose cushions and gradients fractionated from the top. Proteasomes in individual fractions were detected by SDS-PAGE and immunoblotting with anti-FLAG antibody. Sec61p was also detected, and was equal in wild-type and corresponding sec61 mutant membranes (not shown). The positions of proteasomes bound to membranes and of unbound proteasomes are indicated. (D) Dog pancreas ribosomes (1.25 pmol) were bound to 20 eq wild-type or sec61 mutant proteoliposomes under the same conditions as proteasomes above, and analyzed as above using an antibody against the small ribosomal subunit S6. (E) Yeast 19S RP (2 pmol) were bound to 20 eq of the same wild-type and mutant proteoliposomes used for ribosome binding in D, and binding analyzed as in C.

View larger version (44K): [in this window] [in a new window]   Fig. 2. Sec63p and Kar2p co-precipitate with ER-associated proteasomes. (A) Yeast microsomes were isolated from KRY665 in which the Rpt1p subunit of the 19S RP base was tagged with protein A. Membranes were solubilized in DeoxyBigCHAP, and protein-A-tagged Rpt1p and associated proteins isolated by batch absorption to IgG-Sepharose. Protein A-Rpt1p and associated proteins were eluted from washed beads with 0.5 M acetic acid, pH 3.4 (HAc-eluate). Equivalent amounts of microsomes, lysate, material not bound to IgG-Sepharose (supernatant) and 50x HAc-eluate were separated by SDS-PAGE and individual proteins detected by immunoblotting with polyclonal antisera. The asterisk marks a band non-specifically labelled by the Rpt5 antibody. (B) Yeast microsomes were isolated from RJD1171 in which Rpt1p is FLAG tagged. Microsomes were solubilized as above and Rpt1p and associated proteins isolated by adsorption to anti-FLAG agarose. Proteins were eluted with 200 µg/ml FLAG peptide for 1 hour at 4°C. Equivalent amounts of microsomes, lysate, material not bound to anti-FLAG-agarose (supernatant) and 100x of the FLAG eluate were analyzed by SDS-PAGE and immunoblotting as above. The asterisk marks a band non-specifically labelled by the Hrd1 antibody. Note that data in the top and bottom panels are from two separate experiments.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Sec63p does not contribute to proteasome binding to the ER. (A) Yeast 19S RP (2 pmol) were incubated in the absence or presence of 20 eq of SEC63 wild-type or sec63 T652A proteoliposomes as indicated, and binding assessed by flotation in sucrose gradients and SDS-PAGE followed by immunoblotting for the FLAG-tagged Rpn11p subunit as described for Fig. 1. The positions of proteasomes bound to membranes and of unbound proteasomes are indicated. (B) The upper panel shows a schematic drawing of the Sec61 channel and the Sec63 complex; the position of the T652A mutation in the cytosolic domain of Sec63p that disrupts the interaction with Sec62p, is indicated. Membranes from a SEC63 wild-type strain (KRY333) were solubilized in DeoxyBigCHAP and Sec63p depleted by immunoprecipitation, or lysates mock-incubated without antibodies. Depleted and mock-depleted lysates were reconstituted into proteoliposomes, and the degree of depletion of Sec63 and associated proteins assessed by quantitative immunoblotting. (Lower panel) Yeast 19S RP (2 pmol) were incubated with 15 eq of mock-depleted or Sec63p-depleted proteoliposomes as indicated, and binding assessed by flotation in sucrose gradients and SDS-PAGE followed by immunoblotting for the FLAG-tagged Rpt1p subunit.

View larger version (31K): [in this window] [in a new window]   Fig. 4. 19S RP and base compete with each other for binding to the ER. (A) Upper panel: schematic representation of 26S proteasome subparticles. Positions of FLAG tags for purification are indicated; the Pre1p-FLAG strain was used for purification of 20S particles, all other particles were purified from the Rpt1p-FLAG strain RJD1171. Lower panel: Coomassie Blue-stained gel of purified 26S proteasomes and subparticles. Note the slight contamination of the lid fraction with intact 19S RP. (B) Dog PK-RM (10 eq) were incubated with 5 pmol lid or 1 pmol base and analyzed by flotation in a sucrose gradient, followed by SDS-PAGE and immunoblotting for Rpn12 (lid) or Rpt5p (base). (C) Dog PK-RM (10 eq) were incubated with 2.5 pmol base in the absence or presence of 10x excess 19S RP, and analyzed as in B. Base binding was detected using the anti-FLAG antibody, 19S RP binding with anti-Rpn12 antibody, and quantified using chemiluminescence and a CCD camera system (Raytest, Germany).

View larger version (65K): [in this window] [in a new window]   Fig. 5. The 19S RP base in the ATP-bound conformation mediates proteasome binding to the ER. Wild-type 19S RP (1 pmol) or 19S RP with the indicated mutations was incubated with 10 eq dog PK-RM and analyzed by sucrose gradient centrifugation, SDS-PAGE and immunoblotting for the FLAG-tagged Rpn11p subunit. Positions of membrane-bound and unbound 19S RP are indicated.

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