D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p

Abstract

The precise order of molecular events during cell cycle progression depends upon ubiquitin-mediated proteolysis of cell cycle regulators. We demonstrated previously that Hsl1p, a protein kinase that inhibits the Swe1p protein kinase in a bud morphogenesis checkpoint, is targeted for ubiquitin-mediated turnover by the anaphase-promoting complex (APC). Here, we investigate regions of Hsl1p that are critical both for binding to the APC machinery and for APC-mediated degradation. We demonstrate that Hsl1p contains both a destruction box (D box) and a KEN box motif that are necessary for Hsl1p turnover with either APC(Cdc20) or APC(Cdh1). In coimmunoprecipitation studies, the D box of full-length Hsl1p was critical for association with Cdc20p, whereas the KEN box was important for association with Cdh1p. Fusion of a 206-amino-acid fragment of Hsl1p containing these motifs to a heterologous protein resulted in APC-dependent degradation of the fusion protein that required intact D box and KEN box motifs. Finally, this bacterially expressed Hsl1p fusion protein interacted with Cdc20p and Cdh1p either translated in vitro or expressed in and purified from insect cells. Binding to Cdc20p and Cdh1p was disrupted completely by a D box/KEN box double mutant. These results indicate that D box and KEN box motifs are important for direct binding to the APC machinery, leading to ubiquitination and subsequent protein degradation.

Figure 1

Hsl1p has both a D box and a KEN box and both are necessary for APC-mediated degradation in G₁. (A) Hsl1p has a putative KEN box located at amino acids 775–781. The human CDC20 (hCDC20) KEN box and consensus sequence (Pfleger and Kirschner 2000) are shown for comparison. (B) APC^Cdh1-mediated degradation of Hsl1p–HA in G₁-arrested cells requires both a D box and a KEN box. Cells were arrested in G₁ with α-factor (100 ng/mL) and induced to express GAL–HSL1–HA (YJB123 and YJB125), GAL–HSL1^mdb–HA (YJB229 and YJB270), GAL–HSL1^mkb–HA (YJB257 and YJB271) and GAL–HSL1^mdb/mkb–HA (YJB258 and YJB272) by the addition of galactose. Cells were shifted to 37°C to inactivate the APC in cdc23–1 strains. Proteins levels were monitored by immunoblotting with anti-HA antibodies at the indicated times following termination of Hsl1p expression.

Figure 2

Cdc20p can promote the degradation of Hsl1p–HA in G₁. (A) cdh1Δ cdc28-13 cells with (YJB367, YJB368 and YJB380) or without GAL–CDC20–myc (YJB366, YJB377 and YJB379) were arrested in G₁ by inactivation of cdc28-13 at 37°C, then galactose was added to induce expression of GAL–HSL1–HA (YJB366 and YJB367), GAL–HSL1^mdb–HA (YJB377 and YJB378) and GAL–HSL1^mkb–HA (YJB379 and YJB380) and of GAL–CDC20–myc. Levels of wild-type and mutant forms of Hsl1p–HA were monitored at specified times after glucose/cycloheximide addition by quantitative immunoblotting with anti-HA antibodies (see Materials and Methods). Filled and open circles represent Hsl1p–HA without or with Cdc20–myc expression, respectively. The ratios of Hsl1p^mdb–HA (open triangles) and Hsl1p^mkb–HA (open squares) with versus without Cdc20–myc expression are shown. (B) FACS analysis of the indicated strains from the experiment in (A).

Figure 2

Cdc20p can promote the degradation of Hsl1p–HA in G₁. (A) cdh1Δ cdc28-13 cells with (YJB367, YJB368 and YJB380) or without GAL–CDC20–myc (YJB366, YJB377 and YJB379) were arrested in G₁ by inactivation of cdc28-13 at 37°C, then galactose was added to induce expression of GAL–HSL1–HA (YJB366 and YJB367), GAL–HSL1^mdb–HA (YJB377 and YJB378) and GAL–HSL1^mkb–HA (YJB379 and YJB380) and of GAL–CDC20–myc. Levels of wild-type and mutant forms of Hsl1p–HA were monitored at specified times after glucose/cycloheximide addition by quantitative immunoblotting with anti-HA antibodies (see Materials and Methods). Filled and open circles represent Hsl1p–HA without or with Cdc20–myc expression, respectively. The ratios of Hsl1p^mdb–HA (open triangles) and Hsl1p^mkb–HA (open squares) with versus without Cdc20–myc expression are shown. (B) FACS analysis of the indicated strains from the experiment in (A).

Figure 3

Both Cdc20p and Cdh1p can coimmunoprecipitate with APC substrates. (A) Coimmunoprecipitation of GST–Cdc20p and GST–Cdh1p with full-length Hsl1p–HA depends on an intact D box and KEN box, respectively. The different forms of Hsl1p–HA were immunoprecipitated (IP) with anti-HA antibodies from extracts of cells coexpressing either GST–Cdc20p or GST–Cdh1p and then probed for the levels of precipitated Hsl1p–HA proteins (upper) or GST–Cdc20p and GST–Cdh1p (lower) by immunoblot analysis using anti-HA and anti-GST antibodies, respectively. (Lanes 1–10) Strains YJB221, YJB156, YJB230, YJB259, YJB260, YJB222, YJB218, YJB231, YJB261 and YJB262, respectively. (B) GST–Cdc20p and GST–Cdh1p can coimmunoprecipitate with Clb2p–HA. Clb2p–HA was immunoprecipitated with anti-HA antibodies from extracts of cells coexpressing GST–Cdc20p or GST–Cdh1p and proteins were monitored by immunoblot analysis as described in (A). (Lanes 1–6) Strains YJB221, YJB275, YJB276, YJB222, YJB277 and YJB278, respectively. Strains lacking an HA tag (YJB221 and YJB222) but expressing GST–Cdc20p or GST–Cdh1p, respectively, were used as negative controls (No HA) in A and B. mdb, mutant D box; mkb, mutant KEN box.

Figure 4

Analysis of Hsl1p sequences that can act as transposable APC-dependent degradation signals when fused to the carboxyl terminus of the maltose binding protein (MBP). Left panels, schematic representations of amino acids of Hsl1p containing the KEN box and D box motifs (shaded boxes) that were fused to MBP (data not shown). Middle and right panels, stability of MBP–Hsl1p proteins in G₁-arrested cells. Wild-type and cdc23-1 cells were arrested in G₁ with α-factor (100 ng/mL) and then induced to express GAL–MBP–HSL1^667–872 (YJB306 and YJB311), GAL–MBP–HSL1^667–837 (YJB320 and YJB331), GAL–MBP–HSL1^701–872 (YJB321 and YJB335), GAL–MBP–HSL1^701–837 (YJB329 and YJB336), GAL–MBP–HSL1^740–872 (YJB322 and YJB332), GAL–MBP–HSL1^740–837 (YJB330 and YJB337) and GAL–MBP–HSL1^764–872 (YJB307 and YJB311) fusions by the addition of galactose. Strains were subsequently shifted to 37°C to inactivate the APC in the cdc23–1 mutants and the levels of the MBP–Hsl1p fusion proteins were monitored by immunoblot analysis with anti-MBP antibodies at the indicated times after glucose/cycloheximide addition to terminate MBP–Hsl1p synthesis.

Figure 5

Degradation of MBP–Hsl1p^667–872 in G₁ requires intact D box and KEN box motifs. (A) Cells were arrested in G₁ with α-factor and then induced to express WT, mdb, mkb or mdb/mkb isoforms of MBP–Hsl1p^667–872 by the addition of galactose (strains YJB306, YJB326, YJB327 and YJB328, respectively). MBP–Hsl1p^667–872 levels were monitored by immunoblot analysis with anti-MBP antibodies at the indicated times after glucose/cycloheximide addition. (B) D box-dependent binding of GST–Cdc20p to MBP–Hsl1p^667–872. MBP–Hsl1p^667–872 was immunoprecipitated with anti-MBP antibodies from extracts of cells expressing GST–Cdc20p and GST–Cdh1p. Precipitated proteins were analyzed for the presence of GST–Cdc20p and GST–Cdh1p by immunoblot analysis with anti-GST antibodies (lower panels) and for MBP–Hsl1p^667–872 fusion proteins by immunoblot analysis with anti-MBP antibodies (upper panels). (Lanes 1–10) Strains YJB221, YJB309, YJB338, YJB339, YJB340, YJB222, YJB310, YJB341, YJB342 and YJB343, respectively.

Figure 6

Cdc20p and Cdh1p translated in vitro can bind purified MBP–Hsl1p^667–872 in a D box-dependent fashion. E. coli expressed MBP or MBP–Hsl1p^667–872 proteins were purified on amylose resin and then mixed with [³⁵S]methionine-labeled Cdc20p or Cdh1p (see Materials and Methods). Proteins bound to the resin were run on SDS-PAGE and visualized by fluorography (upper panels). Similar amounts of the MBP–Hsl1p^667–872 proteins were present on the amylose beads as visualized by Coomassie staining following SDS-PAGE (lower panels).

Figure 7

Direct binding of Cdh1p to MBP–Hsl1p^667–872. (A) Coomassie stained gels of starting materials used for the direct binding assays in B and in Figure 8. (Left panel) 10 μL of total E. coli extracts expressing MBP or the different forms of MBP–Hsl1p^667–872. (Right panel) 25 μL of cobalt resin from uninfected cell extracts and Cdh1p–6xHis baculovirus-infected Sf9 cell extracts. (B) Cobalt resin was incubated with extracts from insect cells infected with Cdh1p–6xHis baculovirus (lanes1–4) or from uninfected cells (lanes 5–8). Resins were washed and then incubated with 1 mL of the indicated E. coli extracts containing MBP or one of the MBP–Hsl1p^667–872 fusion proteins and washed. Bound proteins were eluted with 150 mM imidizole and visualized by Coomassie staining following SDS-PAGE.

Figure 8

Direct binding between Cdh1p or Cdc20p and MBP–Hsl1p^667–872 depends on D box and KEN box motifs. (A) Coomassie stained gel of binding assay with Cdh1p–6xHis beads (lanes 1–5) and control beads (lanes 6–10). Diluted E. coli extracts containing MBP or MBP–Hsl1p^667–872 proteins were incubated with beads, washed and the bound proteins were eluted with 150 mM imidizole. Asterisks indicate positions of the MBP–Hsl1p^667–872 proteins. (B) Immunoblot analysis of the samples shown in A. One tenth the amount of sample used for Coomassie staining was processed for immunoblot analysis using anti-MBP antibodies. (C) Immunoblot analysis of binding of MBP–Hsl1p^667–872 to GST–Cdc20p beads (lanes 1–5) or uninfected cell beads (lanes 6–10). E. coli extracts were incubated with the glutathione beads as described in (A) except that proteins were not eluted from the beads. Immunoblot analysis was performed with anti-MBP (upper panel) and anti-GST antibodies (lower panel).

Figure 8

Direct binding between Cdh1p or Cdc20p and MBP–Hsl1p^667–872 depends on D box and KEN box motifs. (A) Coomassie stained gel of binding assay with Cdh1p–6xHis beads (lanes 1–5) and control beads (lanes 6–10). Diluted E. coli extracts containing MBP or MBP–Hsl1p^667–872 proteins were incubated with beads, washed and the bound proteins were eluted with 150 mM imidizole. Asterisks indicate positions of the MBP–Hsl1p^667–872 proteins. (B) Immunoblot analysis of the samples shown in A. One tenth the amount of sample used for Coomassie staining was processed for immunoblot analysis using anti-MBP antibodies. (C) Immunoblot analysis of binding of MBP–Hsl1p^667–872 to GST–Cdc20p beads (lanes 1–5) or uninfected cell beads (lanes 6–10). E. coli extracts were incubated with the glutathione beads as described in (A) except that proteins were not eluted from the beads. Immunoblot analysis was performed with anti-MBP (upper panel) and anti-GST antibodies (lower panel).