The Cdc48 ATPase modulates the interaction between two proteolytic factors Ufd2 and Rad23

Abstract

Rad23 and cell division cycle protein 48 (Cdc48), two key regulators of postubiquitylation events, act on distinct and overlapping sets of substrates. The principle underlying their division of labor and cooperation in proteolysis remains elusive. Both Rad23 and Cdc48 bind a ubiquitin protein ligase ubiquitin fusion degradation-2 (Ufd2), and regulate the degradation of Ufd2 substrates. With its ability to bind ubiquitin chains directly and the proteasome via different domains, Rad23 serves as a bridge linking ubiquitylated substrates to the proteasome. The significance and specific role of the Ufd2-Cdc48 interaction are unclear. Here, we demonstrate that mutations in Ufd2 alter its interaction with Cdc48 and impair its function in substrate proteolysis but not in ubiquitylation. Furthermore, Cdc48 promotes the disassembly of the Ufd2-Rad23 complex in an manner that is dependent on ATP and Ufd2 binding, revealing a biochemical role for Cdc48. Rad23 was shown to bind separately to Ufd2 and to the proteasome subunit Rpn1, which define two distinct steps in proteolysis. The action of Cdc48 could free Rad23 from Ufd2 to allow its subsequent association with Rpn1, which in turn may facilitate the orderly transfer of the substrate from the ubiquitylation apparatus to the proteasome.

Fig. 1.

Interactions between Ufd2 and Rad23 or Cdc48. (A) Two-hybrid analysis of the interaction between Ufd2 derivatives and Rad23 or Cdc48. Yeast cells were cotransformed with plasmids encoding the Gal4 DNA-binding domain fused to Ufd2 or its mutant alleles as indicated and the Gal4 activation domain fused to Rad23 or Cdc48. Fourfold dilutions were spotted on the indicated plates. (Right) Growth on the SD-Leu-Trp–His plate is indicative of protein–protein interaction. (Left) Control plate (SD-Leu-Trp) indicates that equal amounts of cells were plated. (B) Purification of Ufd2, Rad23, and Cdc48 from E. coli. A Coomassie blue-stained gel shows purified RGS/His6-tagged Cdc48, Flag-tagged Rad23, and myc-tagged Ufd2 alleles. (C and D) Coimmunoprecipitation analysis of associations between Ufd2 and Cdc48 (C) or Ufd2 and Rad23 (D). Purified Ufd2-myc (2 μg) and RGS/His6-Cdc48 (2 μg) or Flag-Rad23 (2 μg) were mixed in various combinations as indicated in 150 μL binding buffer [50 mM Na-Hepes (pH 7.5), 150 mM NaCl, 5 mM EDTA, 2% Triton X-100, 0.2 mg/mL BSA] containing 1× protease inhibitor mix (Roche) and were incubated with 10 μL (bed volume) of beads coated with various antibodies for 2 h at 4 °C. The beads were washed four times with the binding buffer, followed by SDS/PAGE of the retained proteins and immunoblotting with the antibody indicated.

Fig. 2.

UFD2 mutants impair substrate degradation but not ubiquitylation. (A) Expression of Ufd2 derivatives in yeast. A low-copy plasmid with or without expressing myc-tagged Ufd2 derivatives from the UFD2 promoter was transformed into ufd2Δ cells. Ufd2 was precipitated with myc beads and analyzed by immunoblotting. The amount of Rpt5 in the yeast extracts serves as the loading control. (B) Levels of β-gal activity in ufd2Δ cells carried a plasmid bearing Ub^V76–V–β-gal and a low-copy plasmid with or without expressing UFD2 derivatives from the UFD2 promoter as indicated. The experiments were done more than three times, and average values with SD are shown. (C) UFD substrate Ub^V76–V–β-gal degradation is compromised in ufd2 mutant cells. First, yeast cells containing indicated Ufd2 alleles and a GAL1 promoter-regulated Ub^V76–V–β-gal were grown in raffinose-containing medium. Expression of Ub^V76–V–β-gal was induced by the addition of galactose for 1 h. Samples were taken after promoter shutoff at the time points indicated and analyzed by anti–β-gal Western blot. (D) Quantitation of the data in C for Ub^V76–V–β-gal. SDs are shown. (E) Ufd2 mutants maintain substrate ubiquitylation. Ub^V76–V–β-gal was cotransformed with plasmid p81 bearing Ha-tagged Ub into wild-type or ufd2Δ cells expressing various Ufd2 derivatives. Ub^V76–V–β-gal was isolated with β-gal antibody-coated beads and probed with anti-Ha antibody to detect ubiquitylated Ub^V76–V–β-gal species. Rpt5 serves as a loading control. The UFD2 alleles on the plasmids are labeled, and the UFD2 status in the strains is indicated at the top.

Fig. 3.

Cdc48 promotes the disassembly of the Ufd2–Rad23 complex. (A) Increasing the amount of Cdc48-containing complex destabilizes the Ufd2–Rad23 interaction. The Cdc48-containing complex was purified from Cdc48-TAP strain following a previously described protocol for TAP purification (24). Purified Ufd2-myc (2 μg) and Rad23-Flag (2 μg) (Fig. 1B) were mixed as indicated in 150 μL binding buffer containing ATP and MgCl₂ (36) and were incubated with 10 μL Flag-agarose (Sigma) for 2 h at 4 °C. After the beads were washed two times with the binding buffer, increasing amounts of the Cdc48-containing fraction (5 μg or 10 μg) isolated from the Cdc48-TAP strain were added and incubated for an additional hour (36). The beads were washed three times, followed by SDS/PAGE and immunoblotting with a monoclonal anti-myc antibody. (B and C) The effect of Cdc48 on the Ufd2–Rad23 association requires ATP and Ufd2 binding. The experiments were preformed as described above, except that Ufd2 mutants or ATP analogs (i.e., AMP-PNP, ADP) were used or ATP was omitted in the reaction. Ten micrograms of the Cdc48-containing fraction derived from the Cdc48–TAP complex was used in the experiment. (D) The Cdc48 E588Q mutant defective for ATP hydrolysis is ineffective in promoting Ufd2–Rad23 dissociation. The binding assay was carried out as described above. Wild-type and E588Q Cdc48 tagged with the His6 epitope were purified as described in Fig. 1B. These purified proteins were incubated with yeast extracts to bring down other Cdc48 cofactors in the presence of Ni-NTA beads. The Cdc48-containing complex was eluted with 250 mM imidazole and used in the Ufd2–Rad23 disassembly experiment as in A. Ten micrograms of the Cdc48-containing complex was used in the experiment. (E) Cdc48 does not affect the Rad23–proteasome interaction. Purified Flag-Rad23 was mixed with yeast extracts to bring down the associated proteasome and then was incubated with or without 10 μg of the Cdc48-containing fraction derived from the Cdc48–TAP complex. The Rad23-associated proteasome was detected by anti-Rpt5 antibody.

Fig. 4.

Mutations in Ufd2 alter its binding to Rad23 and Cdc48 in vivo. (A) Coimmunoprecipitation analysis of interactions between Rad23 and Ufd2 derivatives. Proteins were extracted from cells expressing Flag-tagged Rad23 and myc-tagged Ufd2 alleles and immunoprecipitated with beads coupled to various antibodies as indicated. Immunoprecipitates were separated on SDS/PAGE, transferred to a PVDF membrane, and then probed with antibodies. Mutations in Ufd2 are indicated above the panels. The antibodies used for immunoprecipitation (IP) and Western blot (Blot) are indicated at the right of the panels. (B) Coimmunoprecipitation analysis of interactions between Cdc48 and Ufd2 derivatives.

Fig. 5.

Compromised ERAD and UPR activities in ufd2 mutants. (A) Mutations of UFD2 affect the degradation of the ER membrane protein Hmg2. The plasmid expressing wild-type or mutant UFD2 alleles was introduced into ufd2Δ cells harboring myc-Hmg2. Time points were taken after expression shut-off. Membrane proteins were fractioned from these cells and were immunoprecipitated with beads coupled to myc antibody to enrich myc-Hmg2; immunoprecipitates were resolved by SDS/PAGE and then were probed with anti-myc antibody. The stable protein Rpt5 was used as the loading control. (B) Quantitation of the data in A for Hmg2. (C) Elevated UPR activity in cells bearing UFD2 mutants. Yeast cells were transformed with a plasmid that contains the LACZ gene under the control of the promoter of KAR2, which encodes an ER chaperone. Levels of β-gal activity in yeast cells were derived from more than three independent experiments. The strain genotypes are indicated. Bars indicate SD. (D) A model for substrate transfer to the proteasome mediated by a protein–protein interaction network. Cdc48 could play dual roles to promote substrate degradation by assisting Ufd2-mediated ubiquitylation and Rad23-facilitated substrate delivery. The Ub-conjugation phase depicted is based on published evidence (1, 13, 15, 16). The 26S proteasome is drawn as described in ref. with minor modifications. Through interactions with Ufd2, Cdc48, and Rpn1, Rad23 recognizes the substrate and facilitates substrate transfer to the proteasome. See text for details.