Hsl1p, a Swe1p inhibitor, is degraded via the anaphase-promoting complex

Abstract

Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events. The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome. Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates. Hsl1p is absent in G(1), accumulates as cells begin to bud, and disappears in late mitosis. Hsl1p is stabilized by mutations in CDH1 and CDC23, both of which result in compromised APC activity. Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G(1)-arrested cells containing active APC. Mutation of a destruction box motif within Hsl1p (Hsl1p(db-mut)) stabilized Hsl1p. Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies. These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination. Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCF(Met30) in a bud morphogenesis checkpoint. Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events.

FIG. 1

Cdc20p and Cdh1p associate with Hsl1p in cell extracts. Extracts from yeast cells overexpressing Hsl1p-HA, Hsl1p^K110A-HA, or Hsl1p^Δkinase-HA with either GST-Cdc20p, GST-Cdh1p, or GST were incubated with anti-HA antibodies (Santa Cruz Biotechnology) to immunoprecipitate Hsl1p-HA, Hsl1p^K110A-HA, and Hsl1p^Δkinase-HA. Immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis and then immunoblotted with either anti-HA or anti-GST antibodies to detect different forms of Hsl1p-HA (upper panel) or coprecipitation of GST fusion proteins (lower panel). GST-Cdc20p and GST-Cdh1p specifically precipitated with Hsl1p-HA (lanes 1 and 5) and were not precipitated from extracts lacking an HA tag (lanes 4 and 8). Both GST-Cdc20p and GST-Cdh1p coprecipitated with Hsl1p^Δkinase-HA (lanes 3 and 7). No GST-immunoreactive band was detected in the strain expressing GST alone (lane 9). Lanes 1 to 9, strains YJB156, YJB225, YJB223, YJB221, YJB218, YJB226, YJB224, YJB222, and YJB157, respectively.

FIG. 2

Genetic interactions between hsl1Δ and cdc23-1 and between hsl1Δ and cdc28-13. hsl1Δ/HSL1 cdc23-1/CDC23 (YJB131) (A) and hsl1Δ/HSL1 cdc28-13/CDC28 (YJB177) (B) heterozygous diploids were constructed and sporulated (see Materials and Methods). Tetrads were dissected, and the resulting spores were germinated at 23°C. The genotype of each colony was determined by plating onto selective media at 23 or 37°C.

FIG. 3

Micrographs depicting cell morphologies of the indicated haploid strains. (A) Many hsl1Δ cells (strain YJB121) displayed elongated buds. (B) Normal bud morphology could be restored to hsl1Δ cells by a single copy of HSL1-HA (strain YJB168). (C) The hsl1Δ cdc23-1 double mutant (derived from strain YJB131) had elongated buds and formed chains of cells. (D) hsl1Δ cdc28-13 cells (progeny of strain YJB177) were extremely elongated, with an apparent cytokinesis defect. (E and F) Normal cell morphology of the hsl1Δ cdc23-1 and hsl1Δ cdc28-13 double mutants, respectively, was restored by deletion of SWE1 (derived from strain YJB172 and YJB197, respectively). The cells depicted in panels A and B were grown at 30°C, whereas the cells in panels C to F were grown at 23°C. Bar, 4 μm.

FIG. 4

Overexpression of catalytically inactive forms of GAL-HSL1-HA has dominant negative effects. Micrographs of cells overexpressing GAL-HSL1-HA (strain YJB123) and the catalytically inactive forms of HSL1 (strains YJB184 and YJB178) in the presence of endogenous HSL1 are shown. (A and B) Overexpression of GAL-HSL1-HA had no obvious effect on cell morphology, whereas cells overexpressing GAL-HSL1^K110A-HA had elongated buds. (C) Overexpression of GAL-HSL1^Δkinase-HA caused elongated buds and a cytokinesis defect. (D to F) Same strains as in panels A to C but with SWE1 deleted (YJB189, YJB190 and YJB191). Bar, 4 μm.

FIG. 5

Hsl1p-HA levels oscillate during the cell cycle in synchronized cells. Cells of yeast strain YJB90 (cln1,2,3,Δ MET3-CLN2 HSL1-HA) were arrested in G₁ by the addition of methionine to repress CLN2 transcription. When the arrest was complete, a sample was taken for the zero time point and then cells were released from the arrest by resuspension in methionine-free medium. Samples were taken at the indicated times and processed for immunoblot analysis as described in Materials and Methods. (A) Immunoblot of Hsl1p-HA (upper panel) and Mpp10p (lower panel) during the G₁ arrest and release time course. Mpp10p is a nucleolar protein (16) whose levels are constant during the cell cycle. YJB90 cycling cells, molecular mass standards (in kilodaltons), and a strain lacking the HA epitope are shown at right. (B) FACS analysis of each time point to monitor the cell cycle position of the cells throughout the time course.

FIG. 6

Hsl1p-HA has a short half-life in G₁ and is stabilized by mutations that compromise APC activity. (A) Wild-type (strain YJB123), cdc20-1 (strain YJB124), and cdc23-1 (strain YJB125) strains containing an integrated copy of GAL-HSL1-HA were arrested in G₁ with α-factor, induced to express GAL-HSL1-HA by the addition of galactose for 1.5 h, and then shifted to 37°C for 1 h to inactivate cdc20-1 or cdc23-1. The zero time point sample was taken, and then glucose and cycloheximide were added to terminate transcription and translation, respectively. Cell samples were taken at the indicated times and processed for immunoblot analysis as described in Materials and Methods. (B) YJB198 (CDH1 cdc28-13) or YJB199 (cdh1Δ cdc28-13) cells containing GAL-HSL1-HA were arrested in G₁ by shifting the cells to 37°C to inactivate cdc28-13. After 1 h at 37°C, cells were induced to express GAL-HSL1-HA by the addition of galactose and incubated for an additional 2 h at 37°C. A zero time point sample was taken, and then glucose and cycloheximide were added to terminate new synthesis of Hsl1p-HA. Cell samples were taken at the indicated times after glucose and cycloheximide addition. FACS analysis confirmed that cells remained arrested in G₁ at all time points in both panels A and B (data not shown).

FIG. 7

Gin4p-HA and Kcc4p-HA are stable in G₁-arrested cells. Wild-type cells containing an integrated copy of GAL-GIN4-HA (strain YJB247) or GAL-KCC4-HA (strain YJB248) were arrested in G₁ with α-factor and induced to express either gene product by the addition of galactose to the medium for 2.5 h at 30°C. A zero time point sample was taken, and then glucose and cycloheximide were added to terminate new synthesis or Gin4p-HA or Kcc4p-HA. Samples were taken at the indicated time points after glucose and cycloheximide addition.

FIG. 8

Hsl1p-HA degradation and association with Cdc20p and Cdh1p are destruction box dependent. (A) Amino acid sequence of wild-type and Hsl1p^db-mut-HA proteins at residues 828 to 836. Underlined residues represent amino acid changes in the putative destruction box sequence. (B) Hsl1p^db-mut-HA is stabilized in G₁-arrested cells relative to wild-type Hsl1p-HA. Cells containing GAL-HSL1-HA (strain YJB123) or GAL-HSL1^db-mut-HA (strain YJB229) were arrested in G₁ with α-factor and induced to express the different forms of Hsl1p by addition of galactose to the medium for 2.5 h. A zero time point sample was taken, glucose and cycloheximide were added to terminate new synthesis of Hsl1p-HA and Hsl1p^db-mut-HA, and samples were taken at time points as indicated. (C) Hsl1p-HA but not Hsl1p^db-mut-HA can efficiently coimmunoprecipitate GST-Cdc20p and GST-Cdh1p. Hsl1p-HA and Hsl1p^db-mut-HA were immunoprecipitated (IP) from cell extracts coexpressing either GST-Cdc20p or GST-Cdh1p with anti-HA antibodies and then probed for the levels of Hsl1p-HA (upper panels) or GST-Cdc20p or GST-Cdh1p (lower panels) by immunoblot analysis with anti-HA or anti-GST antibodies, respectively.