Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom

Abstract

Background: The nutrient-sensing Tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. Tor is the target of the immunosuppressive drug rapamycin, which in complex with the prolyl isomerase FKBP12 inhibits Tor functions. Rapamycin is a gold standard drug for organ transplant recipients that was approved by the FDA in 1999 and is finding additional clinical indications as a chemotherapeutic and antiproliferative agent. Capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the Tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including Monosiga brevicollis, Salpingoeca rosetta, and Capsaspora owczarzaki.

Results: Interestingly, the Tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. The microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the Tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. Two TOR paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. One such event was identified in the amphibian pathogen Batrachochytrium dendrobatidis, a chytrid responsible for worldwide global amphibian declines and extinctions.

Conclusions: The repeated independent duplications of the TOR gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. These comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments.

Figure 1

The Tor pathway in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. The Tor pathway components investigated in this study in S. cerevisiae (A) and S. pombe (B) are included in this figure. Functional homologs between the two species are indicated in the same shape and color. Sch9, Ypk1, and Gad8 are AGC kinases that are Tor- and PDK-regulated.

Figure 2

Tor protein architecture. Tor protein domain architecture is highly conserved throughout the fungal kingdom. The N-terminal HEAT repeats (blue), the FAT (green) and the FATC (purple) domains participate in protein-protein scaffolding thereby facilitating complex interactions. The FRB domain (yellow) is a highly conserved 100 amino acid sequence necessary for rapamycin interaction. The kinase domain (orange) phosphorylates protein substrates.

Figure 3

The highly conserved FRB domain of Tor. Residues L1971, F1979 and Y2045 are involved in phosphatidic acid binding in mTOR (open arrows). Mutation of S1975 confers rapamycin resistance in mammalian cells, Candida albicans, Cryptococcus neoformans, and Saccharomyces cerevisiae. Residues W2041 and F2048 are required for interaction with rapamycin. All of these amino acid residues are conserved in the species examined in this study. Abbreviations: Sc = Saccharomyces cerevisiae, Sp = Schizosaccharomyces pombe, Po = Pleurotus ostreatus, Mc = Mucor circinelloides, Ro = Rhizopus oryzae, Pb = Phycomyces blakesleeanus, Bd = Batrachochytrium dendrobatidis, Spu = Spizellomyces punctatus.

Figure 4

TOR gene duplication events. Several independent segmental gene duplications or whole genome duplication events have occurred throughout the fungal kingdom resulting in multiple Tor homologs. A whole genome duplication occurred in the Saccharomyces budding yeast lineage prior to the speciation of the sensu stricto, sensu lato, and related Saccharomycotina species. An independent gene duplication event occurred in the Schizosaccharomyces lineage, resulting in 2 Tor homologs in four Schizosaccharomyces fission yeast species. Independent gene duplication events also occurred in Batrachochytrium dendrobatidis and the edible mushroom Pleurotus ostreatus. Numbers at nodes are bootstrap percentages representing 500 replicates.

Figure 5

Synteny analysis of TOR paralogs. Synteny analysis supports the hypothesis that the Tor paralogs in Saccharomyces cerevisiae (A) resulted from a whole genome duplication event, while in Schizosaccharomyces pombe (B) and Batrachochytrium dendrobatidis (C) two Tor paralogs result from independent segmental gene duplication events, and there is no syntenic conservation in the surrounding sequence. Red lines indicate syntenic genes oriented in the same direction whereas blue lines indicate syntenic genes oriented in the opposite direction (i.e., + strand and - strand).