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Analysis of the Cdc28 protein kinase complex by dosage suppression
S I Reed1, J A Hadwiger, H E Richardson
1Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Abstract:
In the interest of identifying components of the Cdc28 protein kinase complex, dosage suppression analysis was performed on temperature-sensitive and dominant negative CDC28 mutations. Dosage suppression is based on a rationale in which elevated expression of wild-type genes can rescue mutations in a target gene as a result of interaction between the respective encoded proteins. Three sequences capable of rescuing a temperature sensitive cdc28 mutation were isolated from a library of wild-type genomic DNA segments in the high copy vector YEp13. Two of these, named CLN1 and CLN2 were found to encode closely related proteins with homology to cyclins. The third, CKS1, encodes an 18K (K = 10(3) Mr) protein that has been shown to be a component of the Cdc28 protein kinase complex and is a homolog of the suc1+ product of fission yeast. A number of dosage suppressors of the CDC28-dn1 dominant negative mutation have been isolated. The one analyzed to date encodes a truncated subunit of the mitochondrial enzyme succinyl-CoA synthetase. The basis for suppression in this case remains to be elucidated.
Insights
Researchers identified new components of the Cdc28 protein kinase complex using dosage suppression. CLN1, CLN2, and CKS1 were found to interact with Cdc28, revealing novel insights into cell cycle regulation.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Cell Cycle Regulation
Background:
- The Cdc28 protein kinase complex is crucial for cell cycle progression in yeast.
- Understanding its components is key to elucidating cell cycle control mechanisms.
Purpose of the Study:
- To identify novel components interacting with the Cdc28 protein kinase complex.
- To characterize genes that can suppress mutations in CDC28.
Main Methods:
- Dosage suppression analysis was performed on temperature-sensitive and dominant-negative CDC28 mutations.
- A genomic DNA library was screened for suppressors of a cdc28 mutation.
- Analysis included isolating and characterizing suppressive DNA segments.
Main Results:
- Three DNA segments, CLN1, CLN2, and CKS1, were identified as suppressors of a temperature-sensitive cdc28 mutation.
- CLN1 and CLN2 encode proteins homologous to cyclins.
- CKS1 encodes an 18 kDa protein, a component of the Cdc28 complex and homolog of fission yeast suc1+.
- A dosage suppressor of the CDC28-dn1 dominant-negative mutation was identified as a truncated subunit of succinyl-CoA synthetase.
Conclusions:
- CLN1, CLN2, and CKS1 are components that interact with the Cdc28 protein kinase complex.
- CKS1 is a functional homolog of the suc1+ gene product.
- Further investigation is needed to understand the mechanism of suppression by the succinyl-CoA synthetase subunit.