Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme.

There is an increasing interest for multifunctional, especially “moonlighting” proteins, defined as proteins endowed with two or more, often unrelated functions associated to a single polypeptide chain1,2. Moonlighting proteins have been documented in a variety of organisms ranging from bacteria and yeast to humans2,3,4,5,6, but not in filamentous fungi so far. The reasons for the growing interest in moonlighting proteins have mainly to do with their inherent biological curiosity, the challenge they pose in genome/proteome annotation and their possible implications in biological circuitry analysis, de novo protein design and human diseases7,8. Multiple, apparently unrelated functions (e.g., cytosolic enzymes also acting as structural, chaperone/scaffold, cell motility-related or transport proteins) have been documented for moonlighting proteins1. In some cases, a strict taxonomic specificity of moonlighting activities has also been documented (see1 for a review). For example, different metabolic enzymes have been recruited as structural proteins of the eye lens (“crystallins”) in a strictly species-specific manner9. A special case in point is represented by metabolic enzymes that moonlight as transcription factors, specifically designated as “trigger enzymes”5 or “metabolism-related transcription factors”10, i.e., proteins with the ability to couple metabolic state sensing with gene expression regulation, thus coordinating cell activity and adaptation in a metabolic signal-dependent manner. The latter include a variety of metabolic enzymes and cofactors (e.g., acetyl-CoA, S-adenosyl methionine and NAD+) directly or indirectly involved in gene expression regulation, with different documented or purported roles such as DNA/RNA binding, modulatory interaction with selected transcription machinery components, co-activator/repressor function and chromatin remodeling11,12,13 (reviewed in10). Following up to the sequencing and annotation of the genome of the black truffle Tuber melanosporum, a filamentous mycorrhizal ascomycetous fungus, we carried out a genome-wide in silico and functional screening of the Tuber proteome searching for proteins endowed with transcription factor (TF) activity14. Given the as yet poor genetic tractability of truffles, this screening, named “transcription activator trap” (TAT)15,16, was conducted in the yeast Saccharomyces cerevisiae. It used as readout the ability of polypeptides representative of the entire Tuber proteome to confer reporter gene transactivation capacity to a deletion derivative of the yeast TF Gal4, capable of DNA-binding but lacking transactivation capacity. In this way, we functionally validated approximately one-fifth (37 out of 201) of the in silico predicted T. melanosporum TFs, but also identified 43 polypeptides whose potential ability to act as transcriptional activators had not been described before. The latter group comprised six metabolic enzymes, with a prevalence of dehydrogenases/reductases. These included PhosphoAdenosine-PhosphoSulfate reductase (PAPS-red), a key enzyme of the sulfur assimilation pathway, responsible for activated sulfate (PAPS) reduction and sulfite formation (see Fig. 1a). Figure 1 Functional and expression characterization of T. melanosporum PAPS reductase. At variance with other fungi, the T. melanosporum genome encodes two PAPS reductase enzymes, designated as PAPS-red A and B. The former enzyme (PAPS-red A), which was retrieved as a potential transcriptional moonlighter in our TAT screen14, is expressed at high levels in fruiting bodies, where sulfur assimilation is also involved in the production of secondary sulfur metabolites (S-Volatile Organic Compounds; S-VOCs) as components of the truffle aroma17. Focusing on this enzyme, we show here that although devoid of a conventional (in silico predictable) nuclear localization signal (NLS), T. melanosporum PAPS-red A has an autonomous nuclear localization capacity. As revealed by functional comparison with PAPS-red enzymes from two unrelated ascomycetes (S. cerevisiae and Neurospora crassa) as well as with the T. melanosporum PAPS-red B enzyme, transcriptional moonlighting appears to be a unique property of PAPS-red A. Transcriptional activation capacity is associated to a transplantable, 23 amino acids C-terminal polypeptide extension, containing a peculiar alternation of hydrophilic and hydrophobic amino acids, as previously observed in several eukaryotic TFs, including yeast Gal418,19. The present work thus identifies a novel transcriptional moonlighting enzyme whose “second job” TF activity may be instrumental to the fine tuning of sulfur metabolism-related genes in an organism with a high reduced-sulfur demand such as T. melanosporum. We also document the potential transcriptional moonlighting activity of six additional black truffle proteins, three of which have an enzymatic metabolic activity as their “first job”.