Evidence for Separation of PRPP Production and Maintenance of Cell Wall Integrity: Two Essential Functions of the PRPP-synthesising Machinery in Saccharomyces cerevisiae

At least 10% of the 6000 genes that make up the genome of Saccharomyces cerevisiae are duplicated. We have identified a family of five PRS genes, each capable of encoding PRPP (phosphoribosyl-pyrophophate) synthetase. The sequence similarity between yeast and human PRS genes has allowed us to create genocopies of the mutations associated with altered Prs activity and examine their effect on yeast physiology. Lithium, a natural Gsk3 inhibitor and a mood stabiliser for the treatment of bipolar disorder inhibits the growth of yeast when PRS1, PRS3 or PRS5 have been deleted indicating an involvement of Prs in neuropathology and cognitive deficits of central nervous system disorders, e. g. Charcot Marie Tooth disease (CMTX5). Prs5 is unusual that it is one of the 11 triply phosphorylated proteins in yeast. Rim11, one of the four Gsk3 paralogous proteins was verified as a partner of Prs5. When the three phosphorylatable S364, S367 and S369 were mutated or deleted the Prs5-Rim11 interaction was reduced by 70% supporting the role of Rim11 as a kinase for posttranslational modification of the three phosphosites in Prs5. This is further supported since Prs5 contains a priming site C-terminal to the site of Gsk3 phosphorylation, a prerequisite of Gsk3 for targeting proteins. Mutation of the three phosphosites of Prs5 impinges on the expression of the transcription factor Rlm1, an endpoint of the cell wall integrity (CWI) pathway. The loss of the heterodimer Prs1/Prs3 also leads to impaired CWI since both interact with Rlm1. The phosphorylated MAP kinase Mpk1/Slt2 of the CWI pathway interacts with Prs1 as shown by immunoprecipitation. In the absence of Prs3, Prs1 is unstable. Prs3 interacts with the kinetochore-associated protein, Nuf2. Prs3 contains a nuclear localisation site whose loss causes caffeine sensitivity and reduction in Rlm1 expression in response to Mpk1/Slt2 activation. Therefore, the synthetic lethality caused by the loss of Prs3 and Prs5 is in fact due to the loss of the Prs1/Prs3 functional subunit and is agreement with three of the five Prs proteins being required to correct CWI signalling by nucleo-cytoplasmic shuttling. We will discuss that the two metabolic functions of Prs, provision of PRPP and maintenance of CWI, is an example of division of labour.