Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation

Abstract

Known activities of the ubiquitin-selective AAA ATPase Cdc48 (p97) require one of the mutually exclusive cofactors Ufd1/Npl4 and Shp1 (p47). Whereas Ufd1/Npl4 recruits Cdc48 to ubiquitylated proteins destined for degradation by the 26S proteasome, the UBX domain protein p47 has so far been linked exclusively to nondegradative Cdc48 functions in membrane fusion processes. Here, we show that all seven UBX domain proteins of Saccharomyces cerevisiae bind to Cdc48, thus constituting an entire new family of Cdc48 cofactors. The two major yeast UBX domain proteins, Shp1 and Ubx2, possess a ubiquitin-binding UBA domain and interact with ubiquitylated proteins in vivo. Deltashp1 and Deltaubx2 strains display defects in the degradation of a ubiquitylated model substrate, are sensitive to various stress conditions and are genetically linked to the 26S proteasome. Our data suggest that Shp1 and Ubx2 are adaptors for Cdc48-dependent protein degradation through the ubiquitin/proteasome pathway.

Figure 1

UBX proteins of S. cerevisiae. UBX (red) and UBA (yellow) domains are labelled. Significant homology outside these domains (Buchberger et al, 2001) is indicated by similar colours. The corresponding open reading frame (ORF) names are: Shp1 (alias Ubx1; YBL058W), Ubx2 (alias Sel1; YML013W), Ubx3 (YDL091C), Ubx4 (YMR067C), Ubx5 (YDR330W), Ubx6 (YJL048C) and Ubx7 (YBR273C).

Figure 2

All yeast members of the UBX family interact with Cdc48. (A) Two-hybrid analysis. Combinations of fusion proteins of the GAL4 activation domain (AD) and DNA-binding domain (BD) with Cdc48 and the indicated UBX proteins were expressed in PJ69-4a. Growth on SC-Leu-Trp-His plates (−His) indicates two-hybrid interaction. (B) Coimmunoprecipitation. Lysates of yeast strains expressing 3HA-epitope-tagged UBX proteins were subjected to anti-HA immunoprecipitation (IP), followed by immunoblotting (WB) using anti-HA and anti-Cdc48 antibodies as indicated. The positions of UBX proteins (asterisks) and Ig heavy chains (HC) are indicated. The parental YPH499 strain served as negative control (WT).

Figure 3

The UBX domain is a key determinant of Cdc48 binding. (A) Two-hybrid interactions between Cdc48 and full-length or C-terminally truncated UBX proteins were analysed as described for Fig 2A. Growth on SC-Leu-Trp-His plates (−His) indicates two-hybrid interaction; and growth on SC-Leu-Trp-Ade plates (−Ade) indicates strong interaction. The C-terminally truncated UBX proteins span the following amino-acid residues: Shp1^ΔUBX, 1–348; Ubx2^ΔUBX, 1–430; Ubx3^ΔUBX, 1–359; Ubx5^ΔUBX, 1–419. (B) Direct binding of isolated UBX domains to Cdc48. In vitro binding of recombinant Cdc48 to beads loaded with GST alone (−) or the indicated GST–UBX domain fusion proteins was detected after SDS–PAGE by staining with Coomassie brilliant blue (CBB; left panel) or anti-Cdc48 western blot (WB; right panel). Equal loading of the beads with GST proteins was confirmed by Coomassie staining (CBB) of the gel (left panel). For the western blot, only 5% of GST–Shp1^UBX, GST–Ubx5^UBX and GST–Ubx7^UBX as compared to GST–Ubx4^UBX and the GST control were loaded to avoid excessive signal strength (right panels). The isolated UBX domains comprise the following residues: Shp1^UBX, 341–423; Ubx4^UBX, 270–358; Ubx5^UBX, 410–500; Ubx7^UBX, 207–295.

Figure 4

UBA/UBX proteins bind ubiquitylated proteins in vivo. Lysates of yeast strains expressing 3HA-epitope-tagged UBA/UBX proteins alone (A), together with a myc-tagged version of ubiquitin (B), or with Ub–P–βGal under the control of a galactose-inducible promoter (C,D), were subjected to immunoprecipitation (IP). (A) Anti-HA IP, followed by WB against ubiquitin (Ub) or HA as indicated. The parental YPH499 strain served as negative control (WT). (B) Anti-myc IP of ubiquitylated proteins from cells expressing myc–ubiquitin (+Ub^myc), followed by WB against myc and HA epitopes. Lysates from strains not expressing myc–ubiquitin (−Ub^myc) served as negative control. The positions of HA-tagged UBX proteins, ubiquitylated proteins (Ub_n) and Ig HCs are indicated. (C) Anti-βGal IP after galactose-induced (Gal) expression of Ub–P–βGal, followed by WB against βGal, Shp1 or HA as indicated. Lysates from glucose-grown cells served as negative control (Glc). (D) Anti-HA IP (HA) after galactose-induced expression of Ub–P–βGal, followed by WB against βGal or HA as indicated. Unspecific immunoglobulins served as negative control (Ig). The positions of HA-tagged UBX proteins, Ub–P–βGal, ubiquitylated Ub–P–βGal (Ub_n–P–βGal), the deubiquitylation product P-βGal and Ig HCs are indicated. A prominent 90 kDa degradation product of Ub-P-βGal is marked by an asterisk. Bands crossreacting with the HA antiserum are marked by closed arrowheads.

Figure 5

Shp1 and Ubx2 are involved in the degradation of Ub-P-βGal. DF5 wild-type (WT) and Δshp1, Δubx2 and ufd1-2 mutant cells expressing Ub-P-βGal (A) or R-βGal (B) were pulse-labelled with [³⁵S]methionine, followed by a chase with excess unlabelled methionine and cycloheximide. After the indicated chase times, βGal was immunoprecipitated and analysed by SDS–PAGE followed by autoradiography. A characteristic degradation product is marked (asterisk). The bottom panels show the quantification by PhosphoImager analysis. In (A), the mean values with standard deviation from three independent experiments are shown.

Figure 6

Shp1 and Ubx2 are linked to stress tolerance and proteasomal degradation. (A) Shp1 and Ubx2 are required for growth under stress conditions. Serial dilutions of WT, Δshp1, Δubx2 and Δrpn10 single mutants, and Δshp1Δrpn10 and Δubx2Δrpn10 double mutants were grown on YPD or SD agar plates containing the indicated additions. (B) Synthetic lethality of Δshp1Δubx2. Upon tetrad dissection, viable Δshp1Δubx2 cells were only obtained if Shp1 (YC-SHP1) or Ubx2 (YC-UBX2) were provided from a plasmid carrying a URA marker (SC-Ura). Forced loss of the plasmid on 5-FOA plates rendered the Δshp1Δubx2 cells inviable.