Identification of novel and conserved functional and structural elements of the G1 cyclin Cln3 important for interactions with the CDK Cdc28 in Saccharomyces cerevisiae.
Regions of the budding yeast G1 cyclin Cln3 were characterized using mutational analysis and viability assays to identify functionally relevant and novel mutant alleles of CLN3. Cyclin proteins are conserved, and Cln3 contains a region with homology to the cyclin box, which is thought to mediate physical interactions with the cyclin-dependent kinase. CLN3 was found to have characteristics similar to the conserved cyclin fold found in higher eukaryotic cyclin boxes, which consist of five alpha-helices. Peptide linker sequences inserted within helices 1, 2, 3 and 5 resulted in a loss of Cln3 function, showing cyclin fold structure similar to that previously observed for the G1 cyclin Cln2. A clustered-charge-to-alanine scan mutagenesis revealed two regions of Cln3 important for Cln3-dependent viability. The first region encompasses the conserved cyclin box. The second region is identified with alanine substitutions located well past the cyclin box, just prior to the C-terminal region of Cln3 important for protein stability. Cln3 with mutational changes in each of these regions are expressed at steady-state levels higher than wild-type Cln3, and show some defect in binding to Cdc28. The conserved hydrophobic patch domain (HPD) of cyclins is present within the first helix of the cyclin box. Alanine substitutions introduced into the HPD of Cln3 and Cln2 show functional defects while maintaining physical interaction with Cdc28 as measured by co-immunoprecipitation assay.
Miller
Mary E
ME
Rhodes College, 2000 North Parkway, Memphis, TN 38112, USA. millerm@rhodes.edu
Cross
Frederick R
FR
Groeger
Alison L
AL
Jameson
Katherine L
KL
D000595:Amino Acid Sequence; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism*; D056746:Cyclin G; D016213:Cyclins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
C079895:CLN3 protein, S cerevisiae; D056746:Cyclin G; D016213:Cyclins; D029701:Saccharomyces cerevisiae Proteins; D017489:CDC28 Protein Kinase, S cerevisiae