1. Volume 42, Issue 5, Pages 637-649 (June 2011)
The Catalytic Activity of Ubp6 Enhances Maturation of the Proteasomal Regulatory Particle 
Eri Sakata, Florian Stengel, Keisuke Fukunaga, Min Zhou, Yasushi Saeki, Friedrich Förster, Wolfgang Baumeister, Keiji Tanaka, Carol V. Robinson 
Molecular Cell 
Volume 42, Issue 5, Pages (June 2011)
DOI: /j.molcel
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2. Molecular Cell 2011 42, 637-649DOI: (10.1016/j.molcel.2011.04.021)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1
Electrospray MS of the Intact 26S Proteasome and Quantitative Proteomics of Assembly Fractions of the 19S RP
(A) Rpn11-3XFLAG 26S proteasome was affinity purified and fractionated by 15%–40% sucrose gradient centrifugation. The purified complex from fraction 16 was analyzed by SDS-PAGE and visualized by protein staining with Coomassie brilliant blue (CBB). SDS-PAGE analysis confirmed the presence of almost equivalent levels of Sem1 with other canonical subunits. The bands corresponding to β7 (∗∗∗), Rpn13 (∗∗), and Sem1 (∗), as identified by MS, are indicated by asterisk.
(B) Fractionated samples were analyzed by SDS-PAGE. The intact 26S proteasome is enriched in fraction 16.
(C) Nanoelectrospray mass spectrum of the intact 26S proteasome. Three prominent charge states series were assigned to the double capped 26S proteasome, the single capped proteasome and the 20S CP, measured as 2,587,352, 1,658,354, and 745,997 Da, respectively.
(D) Subunit composition and relative abundance of the relevant sucrose gradient fractions was analyzed by quantitative proteomics. Intact 26S proteasomes were enriched in f16 after affinity purification with Rpn11-3XFLAG, while the base and 19S were found in f7-9, f10-12 after affinity purification with Rpn1-3XFLAG. Measured peptide intensities were summed and normalized to the protein molecular mass. In addition to the canonical subunits of the proteasome, several PIPs were identified. PIPs are highlighted in orange.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Interactions between the Base and PIPs
(A) Affinity-purified base and 19S complexes via Rpn1-3XFLAG were fractionated by 15%–40% sucrose gradient centrifugation, followed by SDS-PAGE analysis. The base and 19S complexes are enriched in fraction 7–9 and 10–12, respectively.
(B) Predominant charge state series in f7-9 centered at 14,500 m/z with a mass of 659,049 Da, consistent with the assignment [base + Nas6 + Rpn14 + Ubp6] termed the “intact base precursor.” (a) Selection and (b) activation of the complex led to the loss of a highly charged Nas6 and the formation of the corresponding “stripped” complex of [base + Ubp6 + Rpn14]. (c) Spectrum of the intact base without Ubp6. This complex can be measured as 601,839 Da in agreement with the composition [base + Nas6 + Rpn14]. (d) Selection and activation of this complex led to dissociation of Nas6 and the formation of the corresponding “stripped” complex [base + Rpn14] measured as 576,864 Da. See also Figures S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Structural Organization of the Base Complex
(A–C) Addition of DMSO (10% v/v) (A), ammonium hydroxide (10% v/v) (B), and DMSO with additional salt (C) facilitates the dissociation of intact complexes and the formation of subcomplexes. Indicated peaks show identified subcomplexes of the base.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Ubp6 Association with the Rpn1-Rpt2 Assembly Precursor
(A) Fractionated samples from the Rpn1-3XFLAG pull-down were resolved with 4% native-PAGE followed by western blotting with anti-Rpn1 or Ubp6. Ubp6 was present in five bands that contained also Rpn1: the single capped 20S (19S + 20S), the 19S, the base and two smaller subcomplexes. Rpn and Rpt subunits are abbreviated as n and t, respectively.
(B) Identification of components of these precursors. Same samples were analyzed with anti-Rpt1 and Rpt2 antibody.
(C) Double deletions of ubp6 and four chaperones, nas2, nas6, rpn14, and hsm3 exhibit temperature sensitivities. Ten-fold serial dilutions of the indicated strains were spotted onto YPD plate and incubated for 2–3 days at 25°C, 34.5°C, or 37°C. Double deletion of Ubp6 and Hsm3 exhibit the biggest growth defect at 37°C.
(D) Double deletions of Hsm3 and Ubp6 lead to defects in proteasomal assembly. Lysates of deletion mutated cells expressing GFP-labeled Rpn1 (Rpn1-yEGFP) were resolved by native-PAGE. After electrophoresis, the fluorescence of Rpn1-yEGFP was imaged using a Fluoroimager.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 Additional Function of Ubp6 in the Assembly of the Base
(A) Immunoblot analysis of fractions with purified assembly intermediates. Equal amounts of assembly intermediates purified from wild-type or Δubp6 cells were resolved by SDS-PAGE, followed by immunoblotting against ubiquitin antibody. The blots show that ubiquitylated proteins accumulate in the Ubp6 deletion strain.
(B) Growth of wild-type, Δubp6, Δhsm3, double deletions Δubp6Δhsm3, and the double deletions Δubp6Δhsm3 strains expressing Ubp6 (C118A) at different temperatures. Indicated cells were spotted on to YPD plate as in Figure 4C.
(C) Whole-cell extracts from the mutated cells were analyzed by immunoblotting with anti-Ubp6, anti-Ubiquitin (P4D1), and anti-3-Phosphoglycerate kinase (Pgk1) antibodies for their expression levels. Pgk1 served as a loading control.
(D) Recombinant Ubp6 wild-type and catalytic mutant (C118A) were mixed with fractions 7–9 purified from Δubp6 cells, incubated at 37°C and subsequently analyzed by SDS-PAGE/blotting and detected by anti-ubiquitin antibody (P4D1). Ubiquitylated proteins were processed by wild-type Ubp6 but not mutant Ubp6 (C118A).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Pathway of the 26S Proteasome Assembly
The assembly starts with four chaperones, Nas6, Rpn14, Hsm3, and Nas2, binding to specific Rpt subunits. Ubp6 is already associated with Rpn1 of the Hsm3 module in order to trim ubiquitin chains stuck on these precursors and the base. On the way to the mature RP, the chaperones Nas2 and Hsm3 dissociate and the intact base precursor is formed. Binding of Rpn10 allows for the binding of the lid to form the intact 19S RP. At this stage the last chaperones dissociate from the proteasome. During the final steps subsequent binding of the 20S CP leads to the dissociation of the remaining PIPs and the formation of the intact 26S proteasome.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions