Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC

Abstract

Ubiquitin-mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi-subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate-specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co-immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo-related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C-box, a motif required for APC association of Hct1 and conserved among Cdc20-related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.

Fig. 1. Interaction of Hct1 with Clb2 and the APC. Whole-cell extracts (WCE) and proteins immunoprecipitated with antibodies to HA (α-HA IP) or Myc (α-Myc IP) were analysed by immunoblotting with HA- (α-HA), Myc- (α-Μyc) or Cdk1- (α-Cdk1) specific antibodies. (A) Hct1 binds Clb2–Cdk1 complexes. cdc23-1 strains carrying CLB2 or CLB2-myc12 and expressing untagged (–) or HA3-tagged (+) HCT1 from the GAL1 promoter were grown to exponential phase at 25°C and treated with galactose for 2 h. Lane 1, W989; lane 2, W987; lane 3, W1067; lane 4, W1066. (B) Phospho-Hct1 binds Clb2 but not the APC. Wild-type (no myc tag), CDC23-myc9 and CLB2-myc12 strains lacking (–) or containing (+) GALL-HA3-HCT1 were grown to exponential phase (C) or arrested with nocodazole for 3 h (N) and then treated with galactose for 2 h. Lanes 1 and 2, K699, lanes 3 and 4, W1158, lanes 5 and 6, W1641, lanes 7 and 8, W1644, lanes 9 and 10, W1079, lanes 11 and 12, W1156. (C) Hct1 binds Clb2 at normal protein levels. Wild-type strains carrying CLB2 or CLB2-HA3 and untagged (–) or Myc18-tagged (+) HCT1 were arrested with nocodazole for 3 h. Lane 1, K699; lane 2, W2541; lane 3, W9317; lane 4, W2542.

Fig. 1. Interaction of Hct1 with Clb2 and the APC. Whole-cell extracts (WCE) and proteins immunoprecipitated with antibodies to HA (α-HA IP) or Myc (α-Myc IP) were analysed by immunoblotting with HA- (α-HA), Myc- (α-Μyc) or Cdk1- (α-Cdk1) specific antibodies. (A) Hct1 binds Clb2–Cdk1 complexes. cdc23-1 strains carrying CLB2 or CLB2-myc12 and expressing untagged (–) or HA3-tagged (+) HCT1 from the GAL1 promoter were grown to exponential phase at 25°C and treated with galactose for 2 h. Lane 1, W989; lane 2, W987; lane 3, W1067; lane 4, W1066. (B) Phospho-Hct1 binds Clb2 but not the APC. Wild-type (no myc tag), CDC23-myc9 and CLB2-myc12 strains lacking (–) or containing (+) GALL-HA3-HCT1 were grown to exponential phase (C) or arrested with nocodazole for 3 h (N) and then treated with galactose for 2 h. Lanes 1 and 2, K699, lanes 3 and 4, W1158, lanes 5 and 6, W1641, lanes 7 and 8, W1644, lanes 9 and 10, W1079, lanes 11 and 12, W1156. (C) Hct1 binds Clb2 at normal protein levels. Wild-type strains carrying CLB2 or CLB2-HA3 and untagged (–) or Myc18-tagged (+) HCT1 were arrested with nocodazole for 3 h. Lane 1, K699; lane 2, W2541; lane 3, W9317; lane 4, W2542.

Fig. 1. Interaction of Hct1 with Clb2 and the APC. Whole-cell extracts (WCE) and proteins immunoprecipitated with antibodies to HA (α-HA IP) or Myc (α-Myc IP) were analysed by immunoblotting with HA- (α-HA), Myc- (α-Μyc) or Cdk1- (α-Cdk1) specific antibodies. (A) Hct1 binds Clb2–Cdk1 complexes. cdc23-1 strains carrying CLB2 or CLB2-myc12 and expressing untagged (–) or HA3-tagged (+) HCT1 from the GAL1 promoter were grown to exponential phase at 25°C and treated with galactose for 2 h. Lane 1, W989; lane 2, W987; lane 3, W1067; lane 4, W1066. (B) Phospho-Hct1 binds Clb2 but not the APC. Wild-type (no myc tag), CDC23-myc9 and CLB2-myc12 strains lacking (–) or containing (+) GALL-HA3-HCT1 were grown to exponential phase (C) or arrested with nocodazole for 3 h (N) and then treated with galactose for 2 h. Lanes 1 and 2, K699, lanes 3 and 4, W1158, lanes 5 and 6, W1641, lanes 7 and 8, W1644, lanes 9 and 10, W1079, lanes 11 and 12, W1156. (C) Hct1 binds Clb2 at normal protein levels. Wild-type strains carrying CLB2 or CLB2-HA3 and untagged (–) or Myc18-tagged (+) HCT1 were arrested with nocodazole for 3 h. Lane 1, K699; lane 2, W2541; lane 3, W9317; lane 4, W2542.

Fig. 2. Binding of Hct1 and Cdc20 to specific APC substrates. (A) Hct1 binds specific APC substrates. cdc23-1 strains (no HA tag) and cdc23-1 strains carrying GAL1-HA3-CDC5, GAL10-CLB2-HA3, GAL1-CLB3-HA3, GAL1-CLB5-HA3, GAL1-PDS1-HA3 or GAL1-CLN2-HA3 and lacking (–) or containing (+) GAL1-myc3-HCT1 were grown to exponential phase at 25°C and galactose was added for 2 h. Epitope-tagged proteins in whole-cell extracts and in immunoprecipitates prepared with HA antibodies cross-linked to protein A-agarose (α-HA IP) were detected on western blots with HA- (α-HA) or Myc- (α-Myc) specific antibodies. The asterisk in the bottom panel marks a cross-linked antibody species arising from incomplete coupling of antibodies to protein A–agarose. Hct1 coprecipitated with Cdc5 was hyperphosphorylated, indicating that Cdc5 kinase may phosphorylate Hct1. The significance of this hyperphosphorylation is unclear. Lane 1, W1500; lane 2, W1502; lane 3, W1619; lane 4, W1621; lane 5, W1504; lane 6, W1506; lane 7, W1615; lane 8, W1617; lane 9, W1508; lane 10, W1510; lane 11, W1623; lane 12, W1625; lane 13, W1653; lane 14, W1655. (B) Cdc20 but not Hct1-m11 binds Pds1. cdc23-1 strains carrying GAL1-PDS1-HA3 and lacking (–) or containing (+) GAL1-myc3-HCT1-m11 as well as cdc16-1 strains carrying GAL1-PDS1-HA3 and lacking (–) or containing (+) GAL1-myc3-CDC20 were grown to exponential phase at 25°C and treated with galactose for 2 h. Whole-cell extracts and proteins immunoprecipitated with antibodies to Myc (α-Myc IP) were analysed by immunoblotting with HA- (α-HA) or Myc- (α-Myc) specific antibodies. Lane 1, W1623; lane 2, W1741; lane 3, W2538; lane 4, W1588.

Fig. 2. Binding of Hct1 and Cdc20 to specific APC substrates. (A) Hct1 binds specific APC substrates. cdc23-1 strains (no HA tag) and cdc23-1 strains carrying GAL1-HA3-CDC5, GAL10-CLB2-HA3, GAL1-CLB3-HA3, GAL1-CLB5-HA3, GAL1-PDS1-HA3 or GAL1-CLN2-HA3 and lacking (–) or containing (+) GAL1-myc3-HCT1 were grown to exponential phase at 25°C and galactose was added for 2 h. Epitope-tagged proteins in whole-cell extracts and in immunoprecipitates prepared with HA antibodies cross-linked to protein A-agarose (α-HA IP) were detected on western blots with HA- (α-HA) or Myc- (α-Myc) specific antibodies. The asterisk in the bottom panel marks a cross-linked antibody species arising from incomplete coupling of antibodies to protein A–agarose. Hct1 coprecipitated with Cdc5 was hyperphosphorylated, indicating that Cdc5 kinase may phosphorylate Hct1. The significance of this hyperphosphorylation is unclear. Lane 1, W1500; lane 2, W1502; lane 3, W1619; lane 4, W1621; lane 5, W1504; lane 6, W1506; lane 7, W1615; lane 8, W1617; lane 9, W1508; lane 10, W1510; lane 11, W1623; lane 12, W1625; lane 13, W1653; lane 14, W1655. (B) Cdc20 but not Hct1-m11 binds Pds1. cdc23-1 strains carrying GAL1-PDS1-HA3 and lacking (–) or containing (+) GAL1-myc3-HCT1-m11 as well as cdc16-1 strains carrying GAL1-PDS1-HA3 and lacking (–) or containing (+) GAL1-myc3-CDC20 were grown to exponential phase at 25°C and treated with galactose for 2 h. Whole-cell extracts and proteins immunoprecipitated with antibodies to Myc (α-Myc IP) were analysed by immunoblotting with HA- (α-HA) or Myc- (α-Myc) specific antibodies. Lane 1, W1623; lane 2, W1741; lane 3, W2538; lane 4, W1588.

Fig. 3. Binding of Hct1 to derivatives of Clb2 and Cdc5 lacking a destruction box. Whole-cell extracts and proteins immunoprecipitated with antibodies to HA (α-HA IP) or Myc (α-Myc IP) were examined by western analysis using HA- (α-HA), Myc- (α-Myc) or Cdk1- (α-Cdk1) specific antibodies. (A) Hct1 binds Clb2 lacking the destruction box. cdc23-1 strains containing HCT1 or GAL1-myc3-HCT1 and lacking (–) or carrying (+) the indicated GAL10-CLB2-HA3 construct were grown to exponential phase at 25°C and treated with galactose for 1 h. The asterisk marks the IgG heavy chain of the Myc antibody. Lane 1, W1500; lane 2, W1504; lane 3, W1684; lane 4, W1744; lane 5, W1502; lane 6, W1506; lane 7, W1686; lane 8, W1746. (B) Non-phosphorylatable Hct1 binds Clb2 lacking the destruction box. cdc23-1 strains carrying HCT1 or GAL1-myc3-HCT1-m11 and lacking (–) or containing (+) the indicated GAL10-CLB2-HA3 construct were treated as described in (A). Lane 1, W1500; lane 2, W1504; lane 3, W1684; lane 4, W1739; lane 5, W1740; lane 6, W1889. (C) Hct1 binds a truncated version of Cdc5 lacking destruction boxes. cdc23-1 strains carrying HCT1 or GAL1-myc3-HCT1 and lacking (–) or containing (+) the indicated GAL1-CDC5-HA3 construct were treated as described in (A) except that galactose was added for 2 h. Lane 1, W1500; lane 2, W1619; lane 3, W1930; lane 4, W1502; lane 5, W1621; lane 6, W1931.

Fig. 3. Binding of Hct1 to derivatives of Clb2 and Cdc5 lacking a destruction box. Whole-cell extracts and proteins immunoprecipitated with antibodies to HA (α-HA IP) or Myc (α-Myc IP) were examined by western analysis using HA- (α-HA), Myc- (α-Myc) or Cdk1- (α-Cdk1) specific antibodies. (A) Hct1 binds Clb2 lacking the destruction box. cdc23-1 strains containing HCT1 or GAL1-myc3-HCT1 and lacking (–) or carrying (+) the indicated GAL10-CLB2-HA3 construct were grown to exponential phase at 25°C and treated with galactose for 1 h. The asterisk marks the IgG heavy chain of the Myc antibody. Lane 1, W1500; lane 2, W1504; lane 3, W1684; lane 4, W1744; lane 5, W1502; lane 6, W1506; lane 7, W1686; lane 8, W1746. (B) Non-phosphorylatable Hct1 binds Clb2 lacking the destruction box. cdc23-1 strains carrying HCT1 or GAL1-myc3-HCT1-m11 and lacking (–) or containing (+) the indicated GAL10-CLB2-HA3 construct were treated as described in (A). Lane 1, W1500; lane 2, W1504; lane 3, W1684; lane 4, W1739; lane 5, W1740; lane 6, W1889. (C) Hct1 binds a truncated version of Cdc5 lacking destruction boxes. cdc23-1 strains carrying HCT1 or GAL1-myc3-HCT1 and lacking (–) or containing (+) the indicated GAL1-CDC5-HA3 construct were treated as described in (A) except that galactose was added for 2 h. Lane 1, W1500; lane 2, W1619; lane 3, W1930; lane 4, W1502; lane 5, W1621; lane 6, W1931.

Fig. 3. Binding of Hct1 to derivatives of Clb2 and Cdc5 lacking a destruction box. Whole-cell extracts and proteins immunoprecipitated with antibodies to HA (α-HA IP) or Myc (α-Myc IP) were examined by western analysis using HA- (α-HA), Myc- (α-Myc) or Cdk1- (α-Cdk1) specific antibodies. (A) Hct1 binds Clb2 lacking the destruction box. cdc23-1 strains containing HCT1 or GAL1-myc3-HCT1 and lacking (–) or carrying (+) the indicated GAL10-CLB2-HA3 construct were grown to exponential phase at 25°C and treated with galactose for 1 h. The asterisk marks the IgG heavy chain of the Myc antibody. Lane 1, W1500; lane 2, W1504; lane 3, W1684; lane 4, W1744; lane 5, W1502; lane 6, W1506; lane 7, W1686; lane 8, W1746. (B) Non-phosphorylatable Hct1 binds Clb2 lacking the destruction box. cdc23-1 strains carrying HCT1 or GAL1-myc3-HCT1-m11 and lacking (–) or containing (+) the indicated GAL10-CLB2-HA3 construct were treated as described in (A). Lane 1, W1500; lane 2, W1504; lane 3, W1684; lane 4, W1739; lane 5, W1740; lane 6, W1889. (C) Hct1 binds a truncated version of Cdc5 lacking destruction boxes. cdc23-1 strains carrying HCT1 or GAL1-myc3-HCT1 and lacking (–) or containing (+) the indicated GAL1-CDC5-HA3 construct were treated as described in (A) except that galactose was added for 2 h. Lane 1, W1500; lane 2, W1619; lane 3, W1930; lane 4, W1502; lane 5, W1621; lane 6, W1931.

Fig. 4. Stability of Clb2 derivatives in G₁-arrested cells. CLB2-HA3 (W1883), CLB2-Δdb-HA3 (W1884) and CLB2-Δ125-HA3 (W1885) were expressed from the GAL10 promoter in cycling or G₁-arrested (G₁) cdc28-4 bar1-Δ1 cells. Cultures grown at 25°C in raffinose medium were split. One part was further incubated at 25°C (cycling) and induced with galactose for 3 h. The other part was treated with α-factor for 3.5 h and, following arrest in G₁, cultures were shifted to 37°C to inactivate Cdk1 (Cdc28) kinase and then induced with galactose. Samples were taken at the indicated times and the protein levels were determined by immunoblotting with HA-specific antibodies (α-HA). Blots were reprobed with an antiserum to β-tubulin (α-Tub2) as a loading control.

Fig. 5. Sequences of Hct1 required for APC binding. (A) Schematic representation of Hct1 derivatives. Positions of the HA3 epitope, the C-box motif and the seven WD40 repeats are shown. Deleted segments are indicated by a line. Non-functional denotes failure of the overproduced Hct1 derivative to induce Clb2 proteolysis and cell-cycle arrest. (B) The C-box, a conserved motif in proteins of the Cdc20 family. A sequence alignment of the C-box-containing regions of Hct1- and Cdc20-related proteins is shown with residues identical in at least 11 members in bold. A consensus sequence for the C-box is deduced with X indicating any amino acid. Accession numbers and positions of the first amino acids are given. S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces pombe; D.m., Drosophila melanogaster; X.l., Xenopus laevis; M.m., Mus musculus; H.s., Homo sapiens. (C) The C-box of Cdc20 is required for function. We compared a wild-type CDC20 strain (W933) and a temperature sensitive cdc20-3 mutant strain (W1117) transformed with vector alone (pRS316) or with plasmids expressing either CDC20 (pWS923) or CDC20-Δcb (pWS924) from the natural CDC20 promoter. Cells were spotted in serial 10-fold dilutions on plates with glucose-supplemented synthetic medium lacking uracil. Plates were incubated at 25°C for 4 days or at 37°C for 3 days. (D) The C-box of Hct1 is required for function. Upper part: Strains carrying HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) under control of the GAL1 promoter were grown in raffinose medium and treated with nocodazole for 3.5 h. Samples were taken at the indicated times after addition of galactose and protein levels were followed by western analysis with Myc- (α-Myc) and HA- (α-HA) specific antibodies. Blots were reprobed with an antiserum to β-tubulin (α-Tub2) as a loading control. Lower part: We compared a wild-type HCT1 strain (K699) and strains expressing either HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) from the GAL1 promoter. Cells were spotted in serial 10-fold dilutions on XY-plates containing 2% glucose or 2% galactose. Plates were incubated at 25°C for 3 days. (E–G) Interaction of Hct1 derivatives with Clb2 and the APC. Whole-cell extracts and proteins immunoprecipitated with HA- (α-HA IP) or Myc- (α-Myc IP) specific antibodies were analysed by western blotting with the indicated antibodies. (E) Wild-type strains (CLB2) and CLB2-myc12 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were arrested with nocodazole for 3 h and then galactose was added for 2 h. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1079; lane 7, W1156; lane 8, W1760; lane 9, W1761; lane 10, W1842. (F) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were grown to log phase and treated for 2 h with galactose. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1641; lane 7, W1644; lane 8, W1762; lane 9, W1763; lane 10, W1843. (G) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) either the GALL-HA3-HCT1-m11 or GALL-HA3-HCT1-m11-Δcb construct were treated as described in (F). Lane 1, K699; lane 2, W2545; lane 3, W2546; lane 4, W1641; lane 5, W2547; lane 6, W2548.

Fig. 5. Sequences of Hct1 required for APC binding. (A) Schematic representation of Hct1 derivatives. Positions of the HA3 epitope, the C-box motif and the seven WD40 repeats are shown. Deleted segments are indicated by a line. Non-functional denotes failure of the overproduced Hct1 derivative to induce Clb2 proteolysis and cell-cycle arrest. (B) The C-box, a conserved motif in proteins of the Cdc20 family. A sequence alignment of the C-box-containing regions of Hct1- and Cdc20-related proteins is shown with residues identical in at least 11 members in bold. A consensus sequence for the C-box is deduced with X indicating any amino acid. Accession numbers and positions of the first amino acids are given. S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces pombe; D.m., Drosophila melanogaster; X.l., Xenopus laevis; M.m., Mus musculus; H.s., Homo sapiens. (C) The C-box of Cdc20 is required for function. We compared a wild-type CDC20 strain (W933) and a temperature sensitive cdc20-3 mutant strain (W1117) transformed with vector alone (pRS316) or with plasmids expressing either CDC20 (pWS923) or CDC20-Δcb (pWS924) from the natural CDC20 promoter. Cells were spotted in serial 10-fold dilutions on plates with glucose-supplemented synthetic medium lacking uracil. Plates were incubated at 25°C for 4 days or at 37°C for 3 days. (D) The C-box of Hct1 is required for function. Upper part: Strains carrying HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) under control of the GAL1 promoter were grown in raffinose medium and treated with nocodazole for 3.5 h. Samples were taken at the indicated times after addition of galactose and protein levels were followed by western analysis with Myc- (α-Myc) and HA- (α-HA) specific antibodies. Blots were reprobed with an antiserum to β-tubulin (α-Tub2) as a loading control. Lower part: We compared a wild-type HCT1 strain (K699) and strains expressing either HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) from the GAL1 promoter. Cells were spotted in serial 10-fold dilutions on XY-plates containing 2% glucose or 2% galactose. Plates were incubated at 25°C for 3 days. (E–G) Interaction of Hct1 derivatives with Clb2 and the APC. Whole-cell extracts and proteins immunoprecipitated with HA- (α-HA IP) or Myc- (α-Myc IP) specific antibodies were analysed by western blotting with the indicated antibodies. (E) Wild-type strains (CLB2) and CLB2-myc12 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were arrested with nocodazole for 3 h and then galactose was added for 2 h. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1079; lane 7, W1156; lane 8, W1760; lane 9, W1761; lane 10, W1842. (F) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were grown to log phase and treated for 2 h with galactose. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1641; lane 7, W1644; lane 8, W1762; lane 9, W1763; lane 10, W1843. (G) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) either the GALL-HA3-HCT1-m11 or GALL-HA3-HCT1-m11-Δcb construct were treated as described in (F). Lane 1, K699; lane 2, W2545; lane 3, W2546; lane 4, W1641; lane 5, W2547; lane 6, W2548.

Fig. 5. Sequences of Hct1 required for APC binding. (A) Schematic representation of Hct1 derivatives. Positions of the HA3 epitope, the C-box motif and the seven WD40 repeats are shown. Deleted segments are indicated by a line. Non-functional denotes failure of the overproduced Hct1 derivative to induce Clb2 proteolysis and cell-cycle arrest. (B) The C-box, a conserved motif in proteins of the Cdc20 family. A sequence alignment of the C-box-containing regions of Hct1- and Cdc20-related proteins is shown with residues identical in at least 11 members in bold. A consensus sequence for the C-box is deduced with X indicating any amino acid. Accession numbers and positions of the first amino acids are given. S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces pombe; D.m., Drosophila melanogaster; X.l., Xenopus laevis; M.m., Mus musculus; H.s., Homo sapiens. (C) The C-box of Cdc20 is required for function. We compared a wild-type CDC20 strain (W933) and a temperature sensitive cdc20-3 mutant strain (W1117) transformed with vector alone (pRS316) or with plasmids expressing either CDC20 (pWS923) or CDC20-Δcb (pWS924) from the natural CDC20 promoter. Cells were spotted in serial 10-fold dilutions on plates with glucose-supplemented synthetic medium lacking uracil. Plates were incubated at 25°C for 4 days or at 37°C for 3 days. (D) The C-box of Hct1 is required for function. Upper part: Strains carrying HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) under control of the GAL1 promoter were grown in raffinose medium and treated with nocodazole for 3.5 h. Samples were taken at the indicated times after addition of galactose and protein levels were followed by western analysis with Myc- (α-Myc) and HA- (α-HA) specific antibodies. Blots were reprobed with an antiserum to β-tubulin (α-Tub2) as a loading control. Lower part: We compared a wild-type HCT1 strain (K699) and strains expressing either HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) from the GAL1 promoter. Cells were spotted in serial 10-fold dilutions on XY-plates containing 2% glucose or 2% galactose. Plates were incubated at 25°C for 3 days. (E–G) Interaction of Hct1 derivatives with Clb2 and the APC. Whole-cell extracts and proteins immunoprecipitated with HA- (α-HA IP) or Myc- (α-Myc IP) specific antibodies were analysed by western blotting with the indicated antibodies. (E) Wild-type strains (CLB2) and CLB2-myc12 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were arrested with nocodazole for 3 h and then galactose was added for 2 h. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1079; lane 7, W1156; lane 8, W1760; lane 9, W1761; lane 10, W1842. (F) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were grown to log phase and treated for 2 h with galactose. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1641; lane 7, W1644; lane 8, W1762; lane 9, W1763; lane 10, W1843. (G) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) either the GALL-HA3-HCT1-m11 or GALL-HA3-HCT1-m11-Δcb construct were treated as described in (F). Lane 1, K699; lane 2, W2545; lane 3, W2546; lane 4, W1641; lane 5, W2547; lane 6, W2548.

Fig. 5. Sequences of Hct1 required for APC binding. (A) Schematic representation of Hct1 derivatives. Positions of the HA3 epitope, the C-box motif and the seven WD40 repeats are shown. Deleted segments are indicated by a line. Non-functional denotes failure of the overproduced Hct1 derivative to induce Clb2 proteolysis and cell-cycle arrest. (B) The C-box, a conserved motif in proteins of the Cdc20 family. A sequence alignment of the C-box-containing regions of Hct1- and Cdc20-related proteins is shown with residues identical in at least 11 members in bold. A consensus sequence for the C-box is deduced with X indicating any amino acid. Accession numbers and positions of the first amino acids are given. S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces pombe; D.m., Drosophila melanogaster; X.l., Xenopus laevis; M.m., Mus musculus; H.s., Homo sapiens. (C) The C-box of Cdc20 is required for function. We compared a wild-type CDC20 strain (W933) and a temperature sensitive cdc20-3 mutant strain (W1117) transformed with vector alone (pRS316) or with plasmids expressing either CDC20 (pWS923) or CDC20-Δcb (pWS924) from the natural CDC20 promoter. Cells were spotted in serial 10-fold dilutions on plates with glucose-supplemented synthetic medium lacking uracil. Plates were incubated at 25°C for 4 days or at 37°C for 3 days. (D) The C-box of Hct1 is required for function. Upper part: Strains carrying HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) under control of the GAL1 promoter were grown in raffinose medium and treated with nocodazole for 3.5 h. Samples were taken at the indicated times after addition of galactose and protein levels were followed by western analysis with Myc- (α-Myc) and HA- (α-HA) specific antibodies. Blots were reprobed with an antiserum to β-tubulin (α-Tub2) as a loading control. Lower part: We compared a wild-type HCT1 strain (K699) and strains expressing either HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) from the GAL1 promoter. Cells were spotted in serial 10-fold dilutions on XY-plates containing 2% glucose or 2% galactose. Plates were incubated at 25°C for 3 days. (E–G) Interaction of Hct1 derivatives with Clb2 and the APC. Whole-cell extracts and proteins immunoprecipitated with HA- (α-HA IP) or Myc- (α-Myc IP) specific antibodies were analysed by western blotting with the indicated antibodies. (E) Wild-type strains (CLB2) and CLB2-myc12 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were arrested with nocodazole for 3 h and then galactose was added for 2 h. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1079; lane 7, W1156; lane 8, W1760; lane 9, W1761; lane 10, W1842. (F) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were grown to log phase and treated for 2 h with galactose. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1641; lane 7, W1644; lane 8, W1762; lane 9, W1763; lane 10, W1843. (G) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) either the GALL-HA3-HCT1-m11 or GALL-HA3-HCT1-m11-Δcb construct were treated as described in (F). Lane 1, K699; lane 2, W2545; lane 3, W2546; lane 4, W1641; lane 5, W2547; lane 6, W2548.

Fig. 5. Sequences of Hct1 required for APC binding. (A) Schematic representation of Hct1 derivatives. Positions of the HA3 epitope, the C-box motif and the seven WD40 repeats are shown. Deleted segments are indicated by a line. Non-functional denotes failure of the overproduced Hct1 derivative to induce Clb2 proteolysis and cell-cycle arrest. (B) The C-box, a conserved motif in proteins of the Cdc20 family. A sequence alignment of the C-box-containing regions of Hct1- and Cdc20-related proteins is shown with residues identical in at least 11 members in bold. A consensus sequence for the C-box is deduced with X indicating any amino acid. Accession numbers and positions of the first amino acids are given. S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces pombe; D.m., Drosophila melanogaster; X.l., Xenopus laevis; M.m., Mus musculus; H.s., Homo sapiens. (C) The C-box of Cdc20 is required for function. We compared a wild-type CDC20 strain (W933) and a temperature sensitive cdc20-3 mutant strain (W1117) transformed with vector alone (pRS316) or with plasmids expressing either CDC20 (pWS923) or CDC20-Δcb (pWS924) from the natural CDC20 promoter. Cells were spotted in serial 10-fold dilutions on plates with glucose-supplemented synthetic medium lacking uracil. Plates were incubated at 25°C for 4 days or at 37°C for 3 days. (D) The C-box of Hct1 is required for function. Upper part: Strains carrying HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) under control of the GAL1 promoter were grown in raffinose medium and treated with nocodazole for 3.5 h. Samples were taken at the indicated times after addition of galactose and protein levels were followed by western analysis with Myc- (α-Myc) and HA- (α-HA) specific antibodies. Blots were reprobed with an antiserum to β-tubulin (α-Tub2) as a loading control. Lower part: We compared a wild-type HCT1 strain (K699) and strains expressing either HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) from the GAL1 promoter. Cells were spotted in serial 10-fold dilutions on XY-plates containing 2% glucose or 2% galactose. Plates were incubated at 25°C for 3 days. (E–G) Interaction of Hct1 derivatives with Clb2 and the APC. Whole-cell extracts and proteins immunoprecipitated with HA- (α-HA IP) or Myc- (α-Myc IP) specific antibodies were analysed by western blotting with the indicated antibodies. (E) Wild-type strains (CLB2) and CLB2-myc12 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were arrested with nocodazole for 3 h and then galactose was added for 2 h. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1079; lane 7, W1156; lane 8, W1760; lane 9, W1761; lane 10, W1842. (F) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were grown to log phase and treated for 2 h with galactose. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1641; lane 7, W1644; lane 8, W1762; lane 9, W1763; lane 10, W1843. (G) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) either the GALL-HA3-HCT1-m11 or GALL-HA3-HCT1-m11-Δcb construct were treated as described in (F). Lane 1, K699; lane 2, W2545; lane 3, W2546; lane 4, W1641; lane 5, W2547; lane 6, W2548.

Fig. 5. Sequences of Hct1 required for APC binding. (A) Schematic representation of Hct1 derivatives. Positions of the HA3 epitope, the C-box motif and the seven WD40 repeats are shown. Deleted segments are indicated by a line. Non-functional denotes failure of the overproduced Hct1 derivative to induce Clb2 proteolysis and cell-cycle arrest. (B) The C-box, a conserved motif in proteins of the Cdc20 family. A sequence alignment of the C-box-containing regions of Hct1- and Cdc20-related proteins is shown with residues identical in at least 11 members in bold. A consensus sequence for the C-box is deduced with X indicating any amino acid. Accession numbers and positions of the first amino acids are given. S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces pombe; D.m., Drosophila melanogaster; X.l., Xenopus laevis; M.m., Mus musculus; H.s., Homo sapiens. (C) The C-box of Cdc20 is required for function. We compared a wild-type CDC20 strain (W933) and a temperature sensitive cdc20-3 mutant strain (W1117) transformed with vector alone (pRS316) or with plasmids expressing either CDC20 (pWS923) or CDC20-Δcb (pWS924) from the natural CDC20 promoter. Cells were spotted in serial 10-fold dilutions on plates with glucose-supplemented synthetic medium lacking uracil. Plates were incubated at 25°C for 4 days or at 37°C for 3 days. (D) The C-box of Hct1 is required for function. Upper part: Strains carrying HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) under control of the GAL1 promoter were grown in raffinose medium and treated with nocodazole for 3.5 h. Samples were taken at the indicated times after addition of galactose and protein levels were followed by western analysis with Myc- (α-Myc) and HA- (α-HA) specific antibodies. Blots were reprobed with an antiserum to β-tubulin (α-Tub2) as a loading control. Lower part: We compared a wild-type HCT1 strain (K699) and strains expressing either HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) from the GAL1 promoter. Cells were spotted in serial 10-fold dilutions on XY-plates containing 2% glucose or 2% galactose. Plates were incubated at 25°C for 3 days. (E–G) Interaction of Hct1 derivatives with Clb2 and the APC. Whole-cell extracts and proteins immunoprecipitated with HA- (α-HA IP) or Myc- (α-Myc IP) specific antibodies were analysed by western blotting with the indicated antibodies. (E) Wild-type strains (CLB2) and CLB2-myc12 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were arrested with nocodazole for 3 h and then galactose was added for 2 h. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1079; lane 7, W1156; lane 8, W1760; lane 9, W1761; lane 10, W1842. (F) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were grown to log phase and treated for 2 h with galactose. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1641; lane 7, W1644; lane 8, W1762; lane 9, W1763; lane 10, W1843. (G) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) either the GALL-HA3-HCT1-m11 or GALL-HA3-HCT1-m11-Δcb construct were treated as described in (F). Lane 1, K699; lane 2, W2545; lane 3, W2546; lane 4, W1641; lane 5, W2547; lane 6, W2548.

Fig. 5. Sequences of Hct1 required for APC binding. (A) Schematic representation of Hct1 derivatives. Positions of the HA3 epitope, the C-box motif and the seven WD40 repeats are shown. Deleted segments are indicated by a line. Non-functional denotes failure of the overproduced Hct1 derivative to induce Clb2 proteolysis and cell-cycle arrest. (B) The C-box, a conserved motif in proteins of the Cdc20 family. A sequence alignment of the C-box-containing regions of Hct1- and Cdc20-related proteins is shown with residues identical in at least 11 members in bold. A consensus sequence for the C-box is deduced with X indicating any amino acid. Accession numbers and positions of the first amino acids are given. S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces pombe; D.m., Drosophila melanogaster; X.l., Xenopus laevis; M.m., Mus musculus; H.s., Homo sapiens. (C) The C-box of Cdc20 is required for function. We compared a wild-type CDC20 strain (W933) and a temperature sensitive cdc20-3 mutant strain (W1117) transformed with vector alone (pRS316) or with plasmids expressing either CDC20 (pWS923) or CDC20-Δcb (pWS924) from the natural CDC20 promoter. Cells were spotted in serial 10-fold dilutions on plates with glucose-supplemented synthetic medium lacking uracil. Plates were incubated at 25°C for 4 days or at 37°C for 3 days. (D) The C-box of Hct1 is required for function. Upper part: Strains carrying HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) under control of the GAL1 promoter were grown in raffinose medium and treated with nocodazole for 3.5 h. Samples were taken at the indicated times after addition of galactose and protein levels were followed by western analysis with Myc- (α-Myc) and HA- (α-HA) specific antibodies. Blots were reprobed with an antiserum to β-tubulin (α-Tub2) as a loading control. Lower part: We compared a wild-type HCT1 strain (K699) and strains expressing either HA3-HCT1 (W2539) or HA3-HCT1-Δcb (W2540) from the GAL1 promoter. Cells were spotted in serial 10-fold dilutions on XY-plates containing 2% glucose or 2% galactose. Plates were incubated at 25°C for 3 days. (E–G) Interaction of Hct1 derivatives with Clb2 and the APC. Whole-cell extracts and proteins immunoprecipitated with HA- (α-HA IP) or Myc- (α-Myc IP) specific antibodies were analysed by western blotting with the indicated antibodies. (E) Wild-type strains (CLB2) and CLB2-myc12 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were arrested with nocodazole for 3 h and then galactose was added for 2 h. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1079; lane 7, W1156; lane 8, W1760; lane 9, W1761; lane 10, W1842. (F) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) the indicated GALL-HA3-HCT1 construct were grown to log phase and treated for 2 h with galactose. Lane 1, K699; lane 2, W1158; lane 3, W1758; lane 4, W1759; lane 5, W1841; lane 6, W1641; lane 7, W1644; lane 8, W1762; lane 9, W1763; lane 10, W1843. (G) Wild-type strains (CDC23) and CDC23-myc9 strains lacking (–) or containing (+) either the GALL-HA3-HCT1-m11 or GALL-HA3-HCT1-m11-Δcb construct were treated as described in (F). Lane 1, K699; lane 2, W2545; lane 3, W2546; lane 4, W1641; lane 5, W2547; lane 6, W2548.