Determinants of Swe1p degradation in Saccharomyces cerevisiae.
Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCF(Met30) acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCF(Met30)) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist.
McMillan
John N
JN
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Theesfeld
Chandra L
CL
Harrison
Jacob C
JC
Bardes
Elaine S G
ES
Lew
Daniel J
DJ
D000483:Alleles; D000595:Amino Acid Sequence; D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D044783:F-Box Proteins; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011484:Protein-Arginine N-Methyltransferases; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature; D020798:Two-Hybrid System Techniques; D043743:Ubiquitin-Protein Ligase Complexes
D018797:Cell Cycle Proteins; D044783:F-Box Proteins; C470285:MET30 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D011484:Protein-Arginine N-Methyltransferases; C120666:HSL7 protein, S cerevisiae; D043743:Ubiquitin-Protein Ligase Complexes; D011494:Protein Kinases; C099685:SWE1 protein, S cerevisiae; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C081927:CDC5 protein, S cerevisiae; D017489:CDC28 Protein Kinase, S cerevisiae