Unlike most other organisms, the essential five-step Coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the gene(s) encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remains unidentified.Sequence homology analyses suggest three candidates, namely Ykl088w, Hal3 and Vhs3, as putative PPCDC enzymes in Saccharomyces cerevisiae. Interestingly, Hal3 and Vhs3 have been characterized as negative regulatory subunits of the Ppz1 protein phosphatase. Here we show that YKL088w does not encode a third Ppz1 regulatory subunit, and that the essential roles of Ykl088w and the Hal3/Vhs3 pair are complementary, cannot be interchanged and can be attributed to PPCDC-related functions. We demonstrate that while known eukaryotic PPCDCs are homotrimers, the active yeast enzyme is a heterotrimer which consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues.Our results unveil Hal3/Vhs3 as moonlighting proteins, involved in both CoA biosynthesis and protein phosphatase regulation. 3Coenzyme A (CoA, 1) is a ubiquitous and essential cofactor that is utilized by a wide variety of enzymes in reactions where it mainly acts as a carrier and activator of acyl groups 1, 2 . The CoA biosynthetic pathway has been elucidated in various diverse species, including eubacteria (Escherichia coli), plants (Arabidopsis thaliana), and mammals (Homo sapiens) 1,3,4 . These studies have shown that the pathway is universal and consists of the same five enzymatic transformations in all cases, although some diversity exist among the specific proteins that catalyze certain steps 5 . Nonetheless, bioinformatic approaches allow identifying (with a few exceptions 6 ) candidate genes encoding the CoA biosynthetic proteins in nearly all organisms, including Saccharomyces cerevisiae.Interestingly, sequence homology searches suggest three proteins, namely Hal3, Vhs3 and Ykl088w, as candidates that may exhibit phosphopantothenoylcysteine decarboxylase (PPCDC) activity in S. cerevisiae. PPCDC is a flavoprotein that catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine (PPC; 2) to yield 4'-phosphopantetheine (PP; 3), the third step in CoA biosynthesis. While previous studies on PPCDCs have shown some diversity among these enzymes (e.g. the bacterial variants are usually bifunctional proteins that also have phosphopantothenoylcysteine synthetase (PPCS) activity), they all share a common mechanism and active site architecture [7][8][9][10][11] . Furthermore, all PPCDCs characterized so far are monogenic. While the three PPCDC candidates in S. cerevisiae are indeed very similar (Vhs3 and Ykl088w have 49% and 28% sequence identity to the Hal3 protein respectively, see Figure 1a), the identification of Hal3 and Vhs3 as potential PPCDCs is in fact surprising as both proteins have previously been shown to have functions completely unrelated to CoA biosynthesis.Specifically, Hal3 (also known as Sis2) is a conserved protein originally identified as a halotoler...

The Saccharomyces cerevisiae Hal3 protein is a moonlighting protein, able to function both as an inhibitory subunit of the Ppz1 protein phosphatase and as a constituent protomer of an unprecedented heterotrimeric PPCDC (phosphopantothenoylcysteine decarboxylase), the third enzyme of the CoA biosynthetic pathway. In the present study we initiated the dissection of the structural elements required for both disparate cellular tasks by using a combination of biochemical and genetic approaches. We show that the conserved Hal3 core [PD (PPCDC domain)] is necessary for both functions, as determined by in vitro and in vivo assays. The Hal3 NtD (N-terminal domain) is not functional by itself, although in vitro experiments indicate that when this domain is combined with the core it has a relevant function in Hal3's heteromeric PPCDC activity. Both the NtD and the acidic CtD (C-terminal domain) also appear to be important for Hal3's Ppz1 regulatory function, although our results indicate that the CtD fulfils the key role in this regard. Finally, we show that the introduction of two key asparagine and cysteine residues, essential for monofunctional PPCDC activity but absent in Hal3, is not sufficient to convert it into such a homomeric PPCDC, and that additional modifications of Hal3's PD aimed at increasing its resemblance to known PPCDCs also fails to introduce this activity. This suggests that Hal3 has undergone significant evolutionary drift from ancestral PPCDC proteins. Taken together, our work highlights specific structural determinants that could be exploited for full understanding of Hal3's cellular functions.

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.

Saccharomyces cerevisiae Hal3 and Vhs3 are moonlighting proteins, acting both as inhibitors of the serine/threonine protein phosphatase Ppz1 and as subunits (together with Cab3) of the unique heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme of Hemiascomycetous yeast. Both these roles are essential: PPCDC catalyses the third step of coenzyme A biosynthesis, while Ppz1 inhibition is required for regulation of monovalent cation homeostasis. However, the mechanisms by which these proteins’ disparate activities are regulated are not well understood. The PPCDC domains (PDs) of Hal3, Vhs3 and Cab3 constitute the minimum requirement for these proteins to show both PPCDC activity and, in the case of Hal3 and Vhs3, to bind to Ppz1. Using these PD proteins as a model system to study the possibility of dynamic interchange between these roles, we provide evidence that Hal3 binds Ppz1 as a monomer (1:1 stoichiometry), requiring it to de-oligomerize from its usual homo- and heterotrimeric states (the latter having PPCDC activity). This de-oligomerization is made possible by structural features that set Hal3 apart from Vhs3, increasing its ability to undergo monomer exchange. These findings suggest that oligomer interchange may be a significant factor in the functional regulation of these proteins and their various unrelated (moonlighting) functions.

Angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system (RAS), which is primarily responsible for blood pressure homeostasis. Studies have shown that ACE inhibitors yield cardiovascular benefits that cannot be entirely attributed to the inhibition of ACE catalytic activity. It is possible that these benefits are due to interactions between ACE and RAS receptors that mediate the protective arm of the RAS, such as angiotensin II receptor type 2 (ATR) and the receptor MAS. Therefore, in this study, we investigated the molecular interactions of ACE, including ACE homodimerization and heterodimerization with ATR and MAS, respectively. Molecular interactions were assessed by fluorescence resonance energy transfer and bimolecular fluorescence complementation in human embryonic kidney 293 cells and Chinese hamster ovary-K1 cells transfected with vectors encoding fluorophore-tagged proteins. The specificity of dimerization was verified by competition experiments using untagged proteins. These techniques were used to study several potential requirements for the germinal isoform of angiotensin-converting enzyme expressed in the testes (tACE) dimerization as well as the effect of ACE inhibitors on both somatic isoforms of angiotensin-converting enzyme expressed in the testes (sACE) and tACE dimerization. We demonstrated constitutive homodimerization of sACE and of both of its domains separately, as well as heterodimerization of both sACE and tACE with ATR, but not MAS. In addition, we investigated both soluble sACE and the sACE N domain using size-exclusion chromatography-coupled small-angle X-ray scattering and we observed dimers in solution for both forms of the enzyme. Our results suggest that ACE homo- and heterodimerization does occur under physiologic conditions.