THE NH2-TERMINAL EXTENSION OF PROTEIN PHOSPHATASE PPZ1 HAS AN ESSENTIAL FUNCTIONAL-ROLE

Deletion of the yeast Ser/Thr protein phosphatase PPZ1 results in incr eased tolerance to sodium and Lithium. PPZ1 is also important for cell integrity, as ppz1 Delta cells undergo lysis under caffeine stress an d PPZ1 over-expression overrides the lytic defect of mutants in the pr otein kinase C/mitogen-activated protein (MAP) kinase pathway. The PPZ 1 protein can be dissected in two halves. The COOH-terminal half is re lated to type 1 phosphatases, whereas the NH2-terminal half is unrelat ed to phosphatases and contains a consensus site for N-myristoylation. Several mutated versions of PPZ1 have been constructed and tested for complementation of ppz1 Delta mutants. me show that PPZ1 can be myris toylated in vivo and that change of Gly-2 to Ala results in lack of my ristoylation and loss of complementation of salt tolerance, Removal of the entire NH2-terminal half results in complete loss of function, al though it does not abolish the phosphatase activity of the protein exp ressed in Escherichia coli. The deletion of a large region of the NH2- terminal half (residues 17-193) does not affect the ability to complem ent the salt tolerance phenotype but abolish complementation of caffei ne sensitivity, whereas the opposite behavior is observed upon removal of residues from 241 to 318. Mutation of Arg-451 to Leu results in bo th complete loss of function and of phosphatase activity. These result s indicates that the NH2-terminal half of the protein contains structu ral determinants that are specific for certain functions and that the phosphatase activity is required but not sufficient for full PPZ1 func tion.