Abstract
Deletion of the yeast Ser/Thr protein phosphatase PPZ1 results in increased tolerance to sodium and lithium. PPZ1 is also important for cell integrity, as ppz1Delta cells undergo lysis under caffeine stress and PPZ1 overexpression overrides the lytic defect of mutants in the protein kinase C/mitogen-activated protein (MAP) kinase pathway. The PPZ1 protein can be dissected in two halves. The COOH-terminal half is related to type 1 phosphatases, whereas the NH2-terminal half is unrelated to phosphatases and contains a consensus site for N-myristoylation. Several mutated versions of PPZ1 have been constructed and tested for complementation of ppz1Delta mutants. We show that PPZ1 can be myristoylated in vivo and that change of Gly-2 to Ala results in lack of myristoylation and loss of complementation of salt tolerance. Removal of the entire NH2-terminal half results in complete loss of function, although it does not abolish the phosphatase activity of the protein expressed in Escherichia coli. The deletion of a large region of the NH2-terminal half (residues 17-193) does not affect the ability to complement the salt tolerance phenotype but abolish complementation of caffeine sensitivity, whereas the opposite behavior is observed upon removal of residues from 241 to 318. Mutation of Arg-451 to Leu results in both complete loss of function and of phosphatase activity. These results indicates that the NH2-terminal half of the protein contains structural determinants that are specific for certain functions and that the phosphatase activity is required but not sufficient for full PPZ1 function.
Highlights
In addition to the classical Ser/Thr protein phosphatases, namely types 1, 2A, 2B, and 2C [1,2,3], yeast cells contain a number of novel phosphatases
The lytic phenotype is found in mutants in the protein kinase C/MAP1 kinase pathway [9], involved in the proper construction of the yeast cell wall, and there is evidence that the PPZ phosphatases are somehow related to this pathway, since the deletion of PPZ1/PPZ2 is additive to the deletion of the mitogen-activated protein (MAP) kinase gene, and this can be complemented by high copy number expression of both phosphatase genes [9]
Gly-2 Can Be Myristoylated in Vivo and Is Relevant for Function in Salt Tolerance—Since PPZ1 contains a Gly-2 residue that is located within a consensus sequence for N-myristoylation, we explored the possibility that PPZ1 could be myristoylated in vivo and, in this case, if this modification could have functional relevance
Summary
In addition to the classical Ser/Thr protein phosphatases, namely types 1, 2A, 2B, and 2C [1,2,3], yeast cells contain a number of novel phosphatases. The NH2-terminal half of PPZ2 is rich in Ser and Thr residues and, as in PPZ1, contains a consensus sequence for N-myristoylation Strains lacking both PPZ1 and PPZ2 are prone to cell lysis under certain stress situations, such as exposure to caffeine [10] or high temperature [9] and the lytic phenotype can be rescued by addition of 1 M sorbitol to the medium as osmotic cushion. An additional increase is observed when PPZ2 is disrupted in a ppz1⌬ background These mutants display an increased output of sodium and lithium, as a result of an increase in the mRNA levels of the ENA1/PMR2A gene [11], encoding a P-type ATPase that is believed to be the major pump responsible for sodium (and lithium) efflux in yeast cells. Italics denote nucleotides that differs from the wild type sequence and that correspond to the generation of point mutations
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