BTB/POZ Domain Proteins Are Putative Substrate Adaptors for Cullin 3 Ubiquitin Ligases  Rory Geyer, Susan Wee, Scott Anderson, John Yates, Dieter A. Wolf  Molecular Cell  Volume 12, Issue 3, Pages 783-790 (September 2003) DOI: 10.1016/S1097-2765(03)00341-1

Figure 1 BTB/POZ Domain Proteins Interact with Pcu3p (A) Domain structure of S. pombe BTB/POZ domain proteins and their close relatives Skp1p and elongin C (Elc1p). Ank, ankyrin repeats; ATS, α tubulin suppressor. Btb1p was identified as Pcu3p interacting protein by affinity purification and MudPIT. Btb2p and Btb3p were identified in the Sanger Centre S. pombe genome database based on their homology to Btb1p. (B) Lysate from cells carrying endogenously tagged alleles of Pcu3p-13Myc and/or Btb1p-ProA was absorbed to IgG resin, followed by immunoblotting with protein A antibodies to detect Btb1p (top panel), with Myc antibodies to detect Pcu3p (middle panel), and with Pcu1p antisera (bottom panel) as indicated. The left panel shows the total cell lysate before affinity binding to document equal protein input. (C) Lysate from strains carrying endogenously tagged alleles of Pcu3p-13Myc and/or Btb2p-ProA or Btb3p-ProA as indicated was absorbed to IgG resin, followed by immunoblotting. Protein A antibodies were used to detect Btb2p and Btb3p, whereas Myc antibodies were used to detect Pcu3p. The bottom panel shows the total cell lysate before affinity binding. (D) BTB proteins form a complex containing Pcu3p and Pip1p. Cell lysate prepared from the strains of the indicated genotypes was absorbed to IgG resin, followed by immunoblotting with Pcu3p, protein A, or Pip1p antisera. The asterisk denotes crossreactive bands. Btb3p-ProA is visible on the anti-Pcu3p blots (top panels), because the secondary antibody detected the protein A moiety of the fusion protein. (E) Wild-type cells were transformed with a plasmid driving the low level overexpression of N-terminally Myc-tagged Btb2p (pRep81.myc-btb2). Cells were kept under promoter repressive conditions (plus thiamine, off) or in the absence of thiamine to induce expression (on). Cell lysates were immunoprecipitated with Myc antibodies, followed by immunoblotting with anti-Myc, anti-Pcu3p, and anti-Pip1p. The asterisk denotes a band crossreacting with the affinity-purified Pcu3p antisera. Total lysate before immunoprecipitation is shown on the left. Molecular Cell 2003 12, 783-790DOI: (10.1016/S1097-2765(03)00341-1)

Figure 2 BTB/POZ Domains Mediate the Interaction with Pcu3p (A) Wild-type Btb3p (1) and the indicated Btb3p deletion mutants (2–5) were expressed as Myc-tagged proteins in btb3 mutants. Cell lysates were immunoprecipitated with Myc antibodies, and copurification of Pcu3p was determined by immunoblotting with Pcu3p antibodies. The asterisks denote crossreactive bands. Total cell lysates are shown on the bottom. (B) The indicated Btb3p point mutants were expressed as Myc-tagged proteins in btb3 mutants and analyzed for binding of Pcu3p as in (A). The point mutations target residues that are conserved in all S. pombe BTB/POZ domains (see Supplemental Figure S2 at http://www.immunity.com/cgi/content/full/19/3/783/DC1). Molecular Cell 2003 12, 783-790DOI: (10.1016/S1097-2765(03)00341-1)

Figure 3 Control of Btb3p Stability by Pcu3p (A) Wild-type, pcu3, and btb1 deletion strains carrying protein A-tagged Btb3p at the endogenous genomic locus were examined for Btb3p expression levels by immunoblotting with protein A antisera. Tubulin levels are shown as loading control. (B) Both the catalytic and Btb3p binding activities of Pcu3p are required to suppress Btb3p accumulation in pcu3 mutants. Wild-type Pcu3p, and the point mutants Pcu3p-K729K and Pcu3p-Y50G, were expressed at low levels from a pRep81 plasmid in pcu3 deletion mutants carrying protein A tagged Btb3p (two strains each shown). Expression from the plasmids was induced by removal of thiamine (on), and Btb3p expression was assessed by immunoblotting with protein A antibodies. Tubulin is shown as a loading control. (C) Efficient Pcu3p/Btb3p interaction depends on the conserved N-terminal domain of Pcu3p. The N-terminal point mutant Pcu3p-Y50D carries an exchange of a single residue within a conserved region that maps to the same region of CUL1 that mediates its interaction with SKP1/F box adaptor dimers (Zheng et al., 2002). The mutant and wild-type Pcu3p proteins were expressed as Myc-tagged proteins from pRep81 in pcu3 mutants carrying endogenously tagged btb3-proA. Cell lysate was absorbed to IgG beads to pull down Btb3p-proA, followed by immunoblotting with Myc antibodies to detect copurification of Myc-Pcu3p and Myc-Pcu3p-Y50G. Total lysate is shown on the left. (D) Btb3p protein stability in pcu3 mutants. Wild-type or pcu3 deletion strains carrying endogenously tagged Btb3p-ProA (top panels) or Btb1p-ProA (bottom panels) were incubated for the indicated periods in 100 μg/ml cycloheximide (CHX), followed by preparation of cell lysates and detection of Btb1p (lower panel) and Btb3p (upper panel) with protein A antibodies. Tubulin expression is shown as a loading control. The Btb3p blots were additionally examined with the Storm imaging system from Molecular Dynamics. Btb3p band intensities were normalized to tubulin and blotted in the graph shown on the right. (E) Proteasome-dependent ubiquitylation and degradation of Btb3p depends on Pcu3p. Temperature-sensitive mts3-1 proteasome mutants were engineered to carry protein A-tagged Btb3p at the endogenous locus. One strain was also deleted for pcu3, as indicated. Strains were maintained at the permissive temperature of 25°C and then shifted to the restrictive temperature (37°C) for the times indicated. Cell lysates were prepared in a denaturing buffer (see Experimental Procedures) and analyzed by immunoblotting with protein A antibodies to detect Btb3p. The top panel shows a long exposure to visualize low molecular weight species. The bottom panel is a short exposure showing the differences in Btb3p levels in the various strains. Molecular Cell 2003 12, 783-790DOI: (10.1016/S1097-2765(03)00341-1)