Identification of two distinct regions of Net1 in Cdc14 phosphatase regulation in budding yeast

Yu Chung E Chang, Purdue University

Abstract

Cdc14 is a dual specificity phosphatase that is involved in coordinating chromosome segregation and is required for exit from mitosis in Saccharomyces cerevisiae. The activity of yeast Cdc14 is tightly regulated in a cell-cycle dependent manner through an interaction with a multifunctional protein known as Net1. Net1 acts as a potent competitive inhibitor by sequestering Cdc14 in the nucleolus and suppressing its phosphatase activity from G1 phase until early anaphase. In early anaphase the interaction is disrupted and active Cdc14 phosphatase disperses throughout the cell to act on protein substrates. The precise molecular mechanism by which Net1 inhibits Cdc14 has not yet been defined. To gain insight into the mechanism of Cdc14 inhibition we employed site-directed and truncation mutagenesis to identify determinants within Net1 that are required for binding and inhibiting the phosphatase. These Net1 mutants were analyzed for their ability to bind Cdc14 and to inhibit phosphatase activity. The ability of these Net1 mutants to interact with Cdc14 in vivo is also being analyzed. These analyses indicate that Net1 residues 240 to 331 comprise a Cdc14-binding domain. Interestingly, certain truncation mutants failed to inhibit, but retained the capacity to bind Cdc14, suggesting that there are two distinct sites within the Cdc14-binding domain, one that plays a major role in binding but is dispensable for inhibition and another that contains the elements crucial for inhibition. In addition to the biochemical studies with Net1 and Cdc14 complex, structural studies were performed with HMG-CoA synthase. The formation of carbon-carbon bond by Claisen condensation reaction is utilized by various thiolase family enzymes. Among the condensing enzymes, 3-hydroxy-3-methylglutaryl (HMG-CoA) synthase adopts a unique mechanism that activates the methyl group of acetylcysteine to attack acetoacetyl-CoA, whereas in other enzymes the methyl group of acetoacetyl-CoA is activated. This step is essential for the Gram-positive bacteria in the production of isopentenyl diphosphate (IPP), a precursor of isoprenoid in the mevalonate pathway. Mutagenesis studies form G. gallus and E. faecalis HMG-CoA synthase have identified several important catalytic residues (Asp184 and Ser308) that are positioned closely to Glu79 and Ser201. To study the enzymatic involvements of these residues, D184A, D184N, S201A, and S308A mutants were generated and crystallized with acetoacetyl-CoA bound in the active site. In the absence of Ser201 the pantetheine group of aetoacetyl-CoA assumes a different conformation, and in the absence of Ser308 acetoacetyl group of acetoacetyl-CoA assumes a different conformation. Structural analysis of S201A and S308A suggested each serine reside plays a role in the anchoring and stereochemistry determinations of acetoacetyl-CoA. The overall enzymatic reaction of S201A and S308A mutants were comparable to wild type, which suggested the overall enzymatic reaction was not affected by the changes in the conformation of acetoacetyl-CoA. On the other hand, D184N mutant shows no significant changes in the acetoacetyl-CoA position, but the overall enzymatic reaction was completely impaired. Structural analysis indicates the hydrogen bonding network was disrupted between D184N, Tyr236, Ser308, and Gly309. Both Ser308 and Gly309 have been suggested to stabilize the tetrahedral intermediate during the condensation reaction.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Molecular biology

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