Fig 1.
Structure of yeast Rpt subunits and construction of Rpt tet-off strains.
(A) A schematic representation of structural domains of the Rpt subunits. Multiple sequence alignment of yeast Rpt subunits are indicated beneath the domain structure. Rpt1–6 were derived from Saccharomyces cerevisiae. Sequence conservation is indicated beneath the alignment, and conserved residues are marked and color-coded according to the default ClustalX settings. Enlarged sequence alignment is shown in S1 Fig. (B) Structure of 26S proteasome. Molecular surface of the 19S activator particle bound to the 20S core particle (CP; PDB ID 4B4T) (left). The 19S regulatory particle, which contains Rpt AAA+ ATPase subunits (green) and non-ATPase subunits (yellow), caps either end of the 20S CP (gray). Enlarged view of the Rpt AAA+ ATPase subunits are shown as a ribbon (right). N-terminal coiled coils formed by Rpt1–Rpt2 (light red), Rpt4–Rpt5 (red), and Rpt3–Rpt6 (dark red) are colored. Structures are produced by PyMOL. (C) Strain construction by one-step homologous replacement of native promoters with a TetO7-containing cassette. (D) Culture of Rpt tet-off strains was grown to early log phase (OD600 of approximately 0.6–0.8). Ten-fold serial dilutions of these cultures were spotted on YPDA medium agar plates or YPDA medium agar plates containing 10 μg/ml doxycycline. Plates were incubated at 30°C for 3 days and then photographed.
Fig 2.
Expression of mouse and yeast Rpt subunits rescues the growth of yeast with conditional suppression of the wild-type yeast Rpt subunit.
(A) Rpt tet-off strains were transformed with the pAUR123 yeast expression vector encoding yeast (sc) and mouse (m) Rpt subunits, grown to early log phase and individually spotted in duplicate as ten-fold serial dilutions on plates either without (right panel) or with (left panel) doxycycline. Plates were incubated at 30°C for 2 days and then photographed. (B) Multiple sequence alignment of the N-terminal regions of Rpt subunits derived from yeast, worm, fly, frog, and mouse. The coiled-coil regions of Rpt subunits predicted by PairCoil2 are indicated above the sequences (ref. S6 Fig). Sequence conservation is indicated beneath the alignment, and conserved residues are marked and color-coded according to the default ClustalX settings. Multiple sequence alignments of full-length Rpt subunits are shown in S3 Fig.
Fig 3.
Coiled-coil region of Rpt subunits is required for proteasome function.
(A) We constructed a series of deletion mutants (upper figures) in the pAUR123 vector and expressed them in the Rpt tet-off strains. Yeast cultures were grown to early log phase (OD600 of approximately 0.6–0.8). Ten-fold serial dilutions of these cultures were spotted on YPDA medium agar plates or on YPDA medium agar plates containing 10 μg/ml doxycycline (Dox). Plates were incubated at 30°C for 2 days and then photographed (lower panels). (B) Deletion of N-terminal coiled-coil region of Rpt subunits induces the accumulation of polyubiquitinated proteins. Accumulation of polyubiquitinated proteins in the Rpt tet-off yeast cells expressing wild-type and deletion mutants (Rpt1Δ65, Rpt2Δ65, Rpt3Δ65, Rpt4Δ65, Rpt5Δ40, and Rpt6Δ50) were analyzed using western blot with an anti-polyubiquitin antibody. After yeast cells at early log phase were treated with 20 μg/mL Dox for 3 h, cells were harvested and lysed with glass beads in the presence of 10% trichloroacetic acid (TCA) to preserve ubiquitination patterns. PGK1 was used as a loading control.
Fig 4.
Structure-guided mutagenesis of Rpt subunits.
(A) Schematic zenithal representation of two coiled alpha helices. Red, heptad positions a/d; green, g/e; and black, b, c, and f. (B) Coiled-coil-destabilizing (CC−) and coiled-coil-neutral (CC0 and CC00) mutants are grouped as Rpt1–6 subunits. Partial sequences are shown as follows: Rpt1 (40–103); Rpt2 (64–127); Rpt3 (44–107); Rpt4 (50–113); Rpt5 (50–83); and Rpt6 (17–80). The positions of the coiled-coil heptad repeat (abcdefg) are the indicated above sequences.
Fig 5.
Coiled-coil mutations reveal that destabilization of Rpt subunits hampers yeast growth.
(A) Coiled-coil probability for wild-type (black lines) and CC− mutants (blue lines) of Rpt subunits calculated by PairCoil2. The thickness of the red lines represents the confidence of the prediction (p-scores) by Paircoil2. (B) Rpt tet-off strains were transformed with the pAUR123 yeast expression vector encoding wild-type and CC− mutants of Rpt subunits, grown to early log phase, and individually spotted in duplicate as a ten-fold serial dilution on plates either without (right panel) or with (left panel) doxycycline. Plates were incubated at 30°C for 2 days and then photographed. (C) Disruption of the coiled-coil conformation of Rpt subunit induces the accumulation of polyubiquitinated proteins. The accumulation of polyubiquitinated proteins in the Rpt tet-off yeast cells expressing wild-type and CC− mutants (Rpt1CC−, Rpt2CC−, Rpt3CC−, Rpt4CC−, Rpt5CC−, and Rpt6CC−) were analyzed using western blot with an anti-polyubiquitin antibody. After yeast cells at early log phase were treated with 20 μg/mL Dox for 3 h, cells were harvested and lysed with glass beads in the presence of 10% TCA to preserve ubiquitination patterns. PGK1 was used as a loading control.
Fig 6.
Base subcomplex formation by mutant Rpt subunits.
(A) Schematic representation of the TAP procedure used to isolate the assembled base subcomplex from E. coli lysate. (B) Total lysates (top panels) and eluted fraction (middle and bottom panels) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (Top and middle panels) Western blotting analysis: FLAG-tagged Rpt1 (red) and His6-tagged Rpt3 (green) were revealed with anti-FLAG and anti-His6 antibodies, respectively. (Bottom panels) The eluted fraction was separated by SDS-PAGE and visualized using Oriole staining. Deletion mutants (Rpt1Δ65, Rpt2Δ65, Rpt3Δ65, Rpt4Δ65, Rpt5Δ40, and Rpt6Δ50; left panels) and coiled-coil destabilizing (CC−) mutants (Rpt1CC−, Rpt2CC−, Rpt3CC−, Rpt4CC−, Rpt5CC−, and Rpt6CC−; right panels) that caused the defective growth of yeast cells were analyzed as above. In the input of (B), FLAG-Rpt1Δ65 appeared to have lower molecular weight due to its N-terminal deletion because FLAG-tag was attached to Rpt1. With the similar reason, His6-Rpt3Δ65 shows smaller molecular weight. In contrast, His6-Rpt3CC− appeared slightly higher in position than wild-type His6-Rpt3 probably because CC− mutation (Pro replacement) lowered the migration of His6-Rpt3CC−. For the same reason, HA-Rpt3CC− also appeared slightly higher in position than wild-type HA-Rpt3 (S8 Fig).
Fig 7.
Coiled-coil mutations without destabilization of coiled-coils.
(A) Rpt tet-off strains were transformed with the pAUR123 yeast expression vector encoding wild-type and CC0 and CC00 mutants of Rpt subunits, grown to early log phase, and individually spotted in duplicate as a ten-fold serial dilutions on plates either without (right panel) or with (left panel) doxycycline. Plates were incubated at 30°C for 2 days and then photographed. (B) Accumulation of polyubiquitinated proteins in the Rpt tet-off yeast cells expressing wild-type and CC0 or CC00 mutants (Rpt1CC00, Rpt2CC00, Rpt3CC00, Rpt4CC00, Rpt5CC0, and Rpt6CC0) were analyzed using western blot with an anti-polyubiquitin antibody. After the yeast cell at early log phase were treated with 20 μg/mL Dox for 3 h, cells were harvested and lysed with glass beads in the presence of 10% TCA. PGK1 was used as a loading control.