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Evolutionary reshaping of fungal mating pathway scaffold proteins.

ABSTRACT: Scaffold proteins play central roles in the function of many signaling pathways. Among the best-studied examples are the Ste5 and Far1 proteins of the yeast Saccharomyces cerevisiae. These proteins contain three conserved modules, the RING and PH domains, characteristic of some ubiquitin-ligating enzymes, and a vWA domain implicated in protein-protein interactions. In yeast, Ste5p regulates the mating pathway kinases while Far1p coordinates the cellular polarity machinery. Within the fungal lineage, the Basidiomycetes and the Pezizomycetes contain a single Far1-like protein, while several Saccharomycotina species, belonging to the CTG (Candida) clade, contain both a classic Far1-like protein and a Ste5-like protein that lacks the vWA domain. We analyzed the function of C. albicans Ste5p (Cst5p), a member of this class of structurally distinct Ste5 proteins. CST5 is essential for mating and still coordinates the mitogen-activated protein (MAP) kinase (MAPK) cascade elements in the absence of the vWA domain; Cst5p interacts with the MEK kinase (MEKK) C. albicans Ste11p (CaSte11p) and the MAPK Cek1 as well as with the MEK Hst7 in a vWA domain-independent manner. Cst5p can homodimerize, similar to Ste5p, but can also heterodimerize with Far1p, potentially forming heteromeric signaling scaffolds. We found direct binding between the MEKK CaSte11p and the MEK Hst7p that depends on a mobile acidic loop absent from S. cerevisiae Ste11p but related to the Ste7-binding region within the vWA domain of Ste5p. Thus, the fungal lineage has restructured specific scaffolding modules to coordinate the proteins required to direct the gene expression, polarity, and cell cycle regulation essential for mating.

SUBMITTER: Cote P

PROVIDER: S-EPMC3023161 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Evolutionary reshaping of fungal mating pathway scaffold proteins.

Côte Pierre P Sulea Traian T Dignard Daniel D Wu Cunle C Whiteway Malcolm M

mBio 20110111 1

Scaffold proteins play central roles in the function of many signaling pathways. Among the best-studied examples are the Ste5 and Far1 proteins of the yeast Saccharomyces cerevisiae. These proteins contain three conserved modules, the RING and PH domains, characteristic of some ubiquitin-ligating enzymes, and a vWA domain implicated in protein-protein interactions. In yeast, Ste5p regulates the mating pathway kinases while Far1p coordinates the cellular polarity machinery. Within the fungal line ...[more]

PMID: 21249169

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Project description:BACKGROUND: Fungal polyketides include commercially important pharmaceuticals and food additives, e.g. the cholesterol-lowering statins and the red and orange monascus pigments. Presently, production relies on isolation of the compounds from the natural producers, and systems for heterologous production in easily fermentable and genetically engineerable organisms, such as Saccharomyces cerevisiae and Escherichia coli are desirable. Rubrofusarin is an orange polyketide pigment that is a common intermediate in many different fungal biosynthetic pathways. RESULTS: In this study, we established a biosynthetic pathway for rubrofusarin in S. cerevisiae. First, the Fusarium graminearum gene encoding polyketide synthase 12 (PKS12) was heterologously co-expressed with the Aspergillus fumigatus gene encoding phosphopantetheinyl transferase (npgA) resulting in production of YWA1. This aromatic heptaketide intermediate was converted into nor-rubrofusarin upon expression of the dehydratase gene aurZ from the aurofusarin gene cluster of F. graminearum. Final conversion into rubrofusarin was achieved by expression of the O-methyltransferase encoding gene aurJ, also obtained from the aurofusarin gene cluster, resulting in a titer of 1.1 mg/L. Reduced levels of rubrofusarin were detected when expressing PKS12, npgA, and aurJ alone, presumably due to spontaneous conversion of YWA1 to nor-rubrofusarin. However, the co-expression of aurZ resulted in an approx. six-fold increase in rubrofusarin production. CONCLUSIONS: The reconstructed pathway for rubrofusarin in S. cerevisiae allows the production of a core scaffold molecule with a branch-point role in several fungal polyketide pathways, thus paving the way for production of further natural pigments and bioactive molecules. Furthermore, the reconstruction verifies the suggested pathway, and as such, it is the first example of utilizing a synthetic biological "bottom up" approach for the validation of a complex fungal polyketide pathway.

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Evolutionary reshaping of fungal mating pathway scaffold proteins.

Publications

Evolutionary reshaping of fungal mating pathway scaffold proteins.

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