An asymmetric interface between the regulatory and core particles of the proteasome - PubMed (original) (raw)

An asymmetric interface between the regulatory and core particles of the proteasome

Geng Tian et al. Nat Struct Mol Biol. 2011.

Abstract

The Saccharomyces cerevisiae proteasome comprises a 19-subunit regulatory particle and a 28-subunit core particle. To be degraded, substrates must cross the core particle-regulatory particle interface, a site for complex conformational changes and regulatory events. This interface includes two aligned heteromeric rings, one formed by the six ATPase (Rpt) subunits of the regulatory particle and the other by the seven α subunits of the core particle. The Rpt C termini bind to intersubunit cavities in the α-ring, thus directing core particle gating and proteasome assembly. We mapped the Rpt C termini to the α subunit pockets, using a cross-linking approach that revealed an unexpected asymmetry: one side of the ring shows 1:1 contacts of Rpt2-α4, Rpt6-α3 and Rpt3-α2, whereas on the opposite side, the Rpt1, Rpt4 and Rpt5 tails each cross-link to multiple α pockets. Rpt-core particle cross-links are all sensitive to nucleotides, implying that ATP hydrolysis drives dynamic alterations at the core particle-regulatory particle interface.

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Figures

Figure 1

Figure 1

Structural basis for the crosslinking strategy. Detail of a representative α pocket (α4–α5), showing residues used for crosslinking. A surface representation of the α5 subunit is shown along with cartoon representation of the last 12 residues of a PA26 subunit inserted in the α4–α5 pocket. α5 is in purple, α4 in blue. A partial backbone of the α5 subunit is presented in cartoon mode with the side chain of T82 (the residue substituted with Cys and used for crosslinking) and K66 of α5 subunit as well as the C-terminal carbonyl group of PA26 presented in stick mode. The distance between the C-terminus of PA26 and the pocket lysine K66, as well as that between the C-terminus and T82, are labeled (PDB: 1FNT).

Figure 2

Figure 2

Identification of two α–Rpt subunit pairs by cysteine crosslinking. (a,b) Whole cell lysates of yeast were subjected to crosslinking and SDS-PAGE-immunoblot analysis. In each panel, strains bear one α and one Rpt subunit with introduced cysteines. Panels a and b represent α1-I87C and α5-T82C mutants, respectively. Each panel contains a complete set of Rpt C-terminal mutants, as indicated (Rpt1-N467C, Rpt2-L437C, Rpt3-K428C, Rpt4-L437C, Rpt5-A434C, and Rpt6-K405C). A 6xHA tag is present at the C-terminus of each α subunit. BMOE (0.1 mM) is a cysteine-cysteine crosslinker; crosslinking proceeded for 1 hr at 4°C. Crosslinked products are marked by an arrow. The antibody used to probe each panel is indicated at bottom. The electrophoretic mobility and molecular mass (in kDa) of protein standards are indicated at left. (c–f) Purified proteasomes from wild type yeast or mutant yeasts with either a single cysteine substitution or a double cysteine substitution within the two α–Rpt pairs identified in panels a and b were subjected to crosslinking and SDS-PAGE-immunoblot analysis. Panels c and e for α1–Rpt4; panel d and f for α5–Rpt1. Here, as below, proteasomes were purified via a Protein A tag appended to Rpn11(ref. 51).

Figure 3

Figure 3

Identification of the α4–Rpt2 pair. (a) Whole cell lysates from α4-N79C RptX double mutants were subjected to crosslinking and SDS-PAGE-immunoblot analysis. See legend to panel 2a for details. The crosslinked product is marked by an arrow. The antibody used to probe each panel is indicated at bottom. (b,c) Crosslinking of α4 to Rpt2 does not strictly require an introduced cysteine in α4. Purified proteasomes from wild type or mutant yeast with either single or double cysteine substitutions of the Rpt2–α4 pair were crosslinked and subjected to SDS-PAGE-immunoblot analysis. A set of strains in which the α4 subunit was not HA-tagged was used here and the blot was probed with α4-specific antibody. (d) Model of the α3–α4 pocket, showing proximity of endogenous Cys residues (C32 and C46) to the pocket. The surface of α4 is in gray. Cartoon representation of partial backbone of α4 and the C-terminal tail of PA26 are presented in red and green respectively, whereas the pocket surface is highlighted in blue. C32 and C46, along with N79 and C-terminus of PA26, are represented in stick mode and the distances between their β-carbons are labeled (PDB: 1FNT). (e) Whole cell lysates from cells expressing HA-tagged wild type α4 and Cys-substituted Rpt proteins were subjected to crosslinking, followed by SDS-PAGE-immunoblot analysis. See legend to panel 3a for details. (f,g) Purified proteasomes from a set of C32A C46A strains were subjected to crosslinking followed by SDS-PAGE-immunoblot analysis. In lanes marked α4, N79 was substituted with Cys. Those marked Rpt2 are from Rpt2-L437C mutants.

Figure 4

Figure 4

Identification of the α3–Rpt6 and α2–Rpt3 pairs. (a) Whole cell lysates from stationary-phase yeast strains carrying double Cys substitutions as indicated were subjected to crosslinking and SDS-PAGE-immunoblot analysis. All strains expressed α3-T81C. For the Rpt mutant set, see legend to Fig. 2a. Antibody to HA was used to probe for crosslinked products. (b) Whole cell lysates from yeast strains carrying the three identified Rpt–α3 pairs of panel a and in exponential growth (O.D.600=1) were subjected to crosslinking and SDS-PAGE-immunoblot analysis. (c) Purified proteasomes from wild type yeast or mutant yeasts with either a single cysteine substitution or a double cysteine substitution within the α3–Rpt6 pair identified in panels a and b were subjected to crosslinking and SDS-PAGE-immunoblot analysis. (d,e) Purified proteasomes were subjected to crosslinking and SDS-PAGE-immunoblot analysis. Mutant samples were α3-T81C, Rpt6-K405C, and α3-T81C Rpt6-K405C. The blots were probed with antibodies to either HA (to detect α3) or Rpt6. (f,g) Purified proteasomes were subjected to crosslinking and SDS-PAGE-immunoblot analysis. Mutant proteins were α2-A79C, Rpt3-K428C, and α2-A79C Rpt3-K428C. Blots were probed with antibodies to either HA or Rpt3. (g) Similar to Fig. 2a but with purified proteasomes from a set of strains bearing α2-A79C with Rpt C-terminal mutants. The blot was probed for possible crosslinked product using antibodies to Rpt3.

Figure 5

Figure 5

Identification of crosslinks for α5–α6 and α6–α7 pockets. (a) Model of the α6–α7 pocket showing proximity of endogenous residue C113 of α6 to the introduced cysteine (A78C). The two α subunits were painted in magenta and cyan respectively and the C-terminus of PA26 in green. Partial backbones of the α subunits are in cartoon mode. Side chains of the three residues, either with native cysteine or the introduced cysteine, are in stick mode and the distances between their β-carbons are labeled. (b) Whole cell lysates from yeast strains carrying double Cys substitutions as indicated were subjected to crosslinking and SDS-PAGE-immunoblot analysis. All strains expressed α7-I81C with C113 of α6 mutated to alanine. For the Rpt mutants, see legend to Fig.2a. (c,d) Purified proteasomes were subjected to crosslinking and SDS-PAGE-immunoblot analysis. Mutant samples were α7-I81C, Rpt1-N467C, and α7-I81C Rpt1-N467C, all of which are purified from strains with C113 of α6 substituted with alanine. (e,f) As in c and d, except for confirmation of α7-I81C and Rpt5-A434C crosslinking. (g) Similar to a, model of α5–α6 pocket to show proximity of native cysteine C117 in α5 to the position where a cysteine was introduced in α6. α5 was painted in magenta while the others are in the color scheme of a. (h) As in b, crosslinking of whole cell lysates of a set of strain bearing the α6-A78C and α5-C117A mutations. For Rpt mutants, see legend to Fig. 2a. (i–l) Similar as c–f, except that crosslinking was carried out with α6-A78C and Rpt4-L437A or Rpt5-A434C mutants.

Figure 6

Figure 6

Model of the base–CP complex. (a) Proposed model mapping the six Rpt tails to the seven α-pockets of the CP. α subunits are in tan with white inter-subunit pockets. Rpt subunits are in yellow with blue C-terminal tails. Solid arrows from an Rpt’s C-terminal tail represent unique crosslinking between Rpt and specific α pocket. Dashed arrows from the C-terminal tail indicate crosslinking of Rpt to multiple α pockets. (b) Proposed model for the mapping the six Rpt tails into the seven α-pockets of the CP. The α-ring is represented as a molecular surface mode with each subunit in a different color. The pockets formed between subunits are colored black. The six Rpt C-termini are given as white spheres, the positions of which are modeled from the C-termini of the D2 domain of CDC48 (PDB: 3CF1 ref. 54). (c) Ball model of base–CP complex. The C2-fold symmetry axis of the CP lies at the interface between β1 and β1′ as shown. (d) CP-RP misalignment of the proteasome holoenzyme as revealed by cryo-electron microscopy. Averaged images of negatively stained Drosophila melanogaster proteasomes. The density assigned to the Rpt ring is given in orange, the remainder of the proteasome in tan. Adapted with permission from Nickell et al.

Figure 7

Figure 7

Effect of nucleotides on crosslinking between α and Rpt subunits. Proteasomes with introduced cysteines on the nine identified α–Rpt pairs were purified in the presence of 0.1 mM ATP, then incubated at room temperature for 30 min with various nucleotides as indicated at 1 mM, followed by crosslinking at 4°C. Immunoblots of these samples were probed with antibody to HA.

Figure 8

Figure 8

α-subunit N-terminal tails and the Rpt proteins proposed to direct their movements. Top panel as viewed from the RP–CP interface. The closed form of the CP channel is shown. The N-terminal tails (residues 10–18 of α1, 1–11 of α2, 2–12 of α3, 3–10 of α4, 9–14 of α5, 2–12 of α6, and 4–13 of α7) are shown in cartoon mode and painted in different colors. The remaining residues are in surface representation, colored in grey. The α carbon of the last residue of each N-terminal tail is in sphere mode. The bottom panel is a side view of the α-ring gate with the same color scheme as at top. Note that Rpt4 and Rpt6 are positioned to disrupt the N-terminal tails of α7 and α2, but do not open the gate, consistent with their lack of a HbYX motif and the minor role in gating played by the peripheral α7 N-terminus.

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