Moonlighting proteins Hal3 and Vhs3 form a heteromeric PPCDC with Ykl088w in yeast CoA biosynthesis

Abstract

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...

Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans

“…We demonstrated in biochemical assays that, in agreement with the structural conservation of its catalytic domain, the CaPpz1 protein exhibits phosphatase activity. This activity was inhibited by the ScHal3 protein, which is a specific inhibitor of PPZ phosphatases and, at the same time, one of the putative subunits of phosphopantothenoylcysteine decarboxylase (Ruiz et al, 2009). It should be noted that in C. albicans there are two distantly related Hal3 orthologues (orf19.7378 and orf19.3260).…”

The Expanding Landscape of Moonlighting Proteins in Yeasts

“…In the yeast group derived from an ancestor with a duplicated genome (102,103), to which S. cerevisiae belongs, the enzyme would be formed by one Cab3 subunit plus two units of Hal3 and/or Vhs3. In archiascomycetaceous yeasts, to which S. pombe belongs, a homotrimeric protein formed by a Hal3-like protein is found (93). This diversity is conditioned by the structure of the active enzyme.…”

“…The proteins of S. cerevisiae that participate in the decarboxylation of phosphopantothenoyl-L-cysteine to pantotheine-4-phosphate, one of the steps in the biosynthetic pathway of CoA from pantothenate, have been identified in the first decade of the 21st century (93). A heterotrimer formed by the protein Cab3 and two monomers that can be either Hal3 or Vhs3 is required to catalyze that reaction in budding yeast.…”

The tricks plants use to reach appropriate light

“…HAL3 is now known to function as a 4′-phosphopantothenoylcysteine (PPC) decarboxylase [133,134,[136][137][138] or a subunit of PPC decarboxylase [139]. However, studies on modified HAL3 and complementary experiments have indicated that its role in promoting the cell cycle is independent of its function in catalyzing PPC [126].…”