The Schizosaccharomyces pombe fusion gene hal3 encodes three distinct activities

Abstract

SummarySaccharomyces cerevisiae Hal3 and Vhs3 are moonlighting proteins, forming an atypical heterotrimeric decarboxylase (PPCDC) required for CoA biosynthesis, and regulating cation homeostasis by inhibition of the Ppz1 phosphatase. The Schizosaccharomyces pombe ORF SPAC15E1.04 (renamed as Sp hal3) encodes a protein whose amino-terminal half is similar to Sc Hal3 whereas its carboxyl-terminal half is related to thymidylate synthase (TS). We show that Sp Hal3 and/or its N-terminal domain retain the ability to bind to and modestly inhibit in vitro S. cerevisiae Ppz1 as well as its S. pombe homolog Pzh1, and also exhibit PPCDC activity in vitro and provide PPCDC function in vivo, indicating that Sp Hal3 is a monogenic PPCDC in fission yeast. Whereas the Sp Hal3 N-terminal domain partially mimics Sc Hal3 functions, the entire protein and its carboxyl-terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function. Additionally, we show that the 70 kDa Sp Hal3 protein is not proteolytically processed under diverse forms of stress and that, as predicted, Sp hal3 is an essential gene. Therefore, Sp hal3 represents a fusion event that joined three different functional activities in the same gene. The possible advantage derived from this surprising combination of essential proteins is discussed.

The Expanding Landscape of Moonlighting Proteins in Yeasts

“…Surprisingly, this gene also showed a 3= fragment with sequence homology to thymidylate synthase-encoding genes. The authors resequenced the genomic locus of S. pombe and that of the related yeast Schizosaccharomyces japonicus and confirmed the unusual structure of the gene in both cases (99). The same authors also showed that the S. pombe Hal3 protein or even its N-terminal domain could rescue an S. cerevisiae cab3 single mutant or hal3 vhs3 double mutant, indicating that this protein is sufficient to achieve PPCDC activity.…”

“…In an interesting evolutionary study, the same group examined the properties of the corresponding protein in the fission yeast S. pombe (99). Although there has been some controversy about the precise location of S. pombe on the fungal evolutionary tree (100), it is clear that the ancestors of S. cerevisiae and S. pombe separated quite early, with estimations ranging between 420 and 330 million years ago (101).…”

“…The protein was also able to inhibit the in vitro activity of S. cerevisiae Ppz1 or the corresponding S. pombe homolog Pzh1. In addition, Hal3 from S. pombe or its C-terminal domain allowed the growth of a cdc21 mutant of S. cerevisiae (99) whose phenotype is due to a lack of thymidylate synthase. Thus, Hal3 from S. pombe has moonlighting activity and is also a "bifunctional" enzyme that acquired a completely unrelated activity, probably by gene fusion; according to Molero et al (99), such fusion events are rare in S. pombe, and only 12 were found by using a dedicated algorithm.…”

Characterization of the atypical Ppz/Hal3 phosphatase system from the pathogenic fungus Cryptococcus neoformans

“…The visible-UV absorption profile of recombinant CnHal3a and CnHal3b devoid of the GST tag was obtained in a Nanodrop ND1000. Cross-linking of recombinant CnHal3b was carried out as in (Molero et al , 2013), except that 0.01% glutaraldehyde was used. Evaluation of disordered regions in the Cryptococcus proteins was done using the Genesilico MetaDisorder server (Kozlowski and Bujnicki, 2012) and the IUPred software (Dosztanyi et al , 2005).…”

“…However, whereas both CaHal3 and CaCab3 retained their predicted PPCDC‐related functions, only CaCab3 exhibited phosphatase regulatory potential in vivo, suggesting that in C. albicans CaCab3, but not CaHal3, acts as a moonlighting protein (Petrényi et al , ). Other fungal genomes present interesting structural alternatives, such as that of fission yeast, in which a likely gene fusion event generated a single gene product (SpHal3), composed of an N‐terminal PPCDC core, including a short acidic tail, followed by a functional C‐terminal thymidylate synthase domain (Molero et al , ). SpHal3 is a functional PPCDC but exhibits limited capacity to inhibit ScPpz1 or the S. pombe homolog Pzh1 in vitro .…”