Gliotoxin, and other related molecules, are encoded by multi-gene clusters and biosynthesized by fungi using non-ribosomal biosynthetic mechanisms. Almost universally described in terms of its toxicity towards mammalian cells, gliotoxin has come to be considered as a component of the virulence arsenal of Aspergillus fumigatus. Here we show that deletion of a single gene, gliT, in the gliotoxin biosynthetic cluster of two A. fumigatus strains, rendered the organism highly sensitive to exogenous gliotoxin and completely disrupted gliotoxin secretion. Addition of glutathione to both A. fumigatus ΔgliT strains relieved gliotoxin inhibition. Moreover, expression of gliT appears to be independently regulated compared to all other cluster components and is up-regulated by exogenous gliotoxin presence, at both the transcript and protein level. Upon gliotoxin exposure, gliT is also expressed in A. fumigatus ΔgliZ, which cannot express any other genes in the gliotoxin biosynthetic cluster, indicating that gliT is primarily responsible for protecting this strain against exogenous gliotoxin. GliT exhibits a gliotoxin reductase activity up to 9 µM gliotoxin and appears to prevent irreversible depletion of intracellular glutathione stores by reduction of the oxidized form of gliotoxin. Cross-species resistance to exogenous gliotoxin is acquired by A. nidulans and Saccharomyces cerevisiae, respectively, when transformed with gliT. We hypothesise that the primary role of gliotoxin may be as an antioxidant and that in addition to GliT functionality, gliotoxin secretion may be a component of an auto-protective mechanism, deployed by A. fumigatus to protect itself against this potent biomolecule. The pathogenic fungus Aspergillus fumigatus causes disease in immunocompromised individuals such as cancer patients. The fungus makes a small molecule called gliotoxin which helps A. fumigatus bypass the immune system in ill people, and cause disease. Although a small molecule, gliotoxin biosynthesis is enabled by a complex series of enzymes, one of which is called GliT, in A. fumigatus. Amazingly, nobody has really considered that gliotoxin might be toxic to A. fumigatus itself. Here we show that absence of GliT makes A. fumigatus highly sensitive to added gliotoxin and inhibits fungal growth, both of which can be reversed by restoring GliT. Neither can the fungus make or release its own gliotoxin when GliT is missing. We also show that gliotoxin sensitivity can be totally overcome by adding glutathione, which is an important anti-oxidant within cells. We demonstrate that gliotoxin addition increases the production of GliT, and that GliT breaks the disulphide bond in gliotoxin which may be a step in the pathway for gliotoxin protection or release from A. fumigatus. We conclude that gliotoxin may mainly be involved in protecting A. fumigatus against oxidative stress and that it is an accidental toxin.

In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity.

Inactivation of Hsp104 by guanidine is contended to be the mechanism by which guanidine cures yeast prions. We now find an Hsp104 mutation (D184N) that confers resistance to guanidine-curing of the yeast [ PSI ⁺ ] prion. In an independent screen we isolated an HSP104 allele altered in the same residue (D184Y) that dramatically impairs [ PSI ⁺ ] propagation in a temperature-dependent manner. Directed mutagenesis of HSP104 produced additional alleles that conferred varying degrees of resistance to guanidine-curing or impaired [ PSI ⁺ ] propagation. The mutations similarly affected propagation of the [ URE3 ] prion. Basal and induced abundance of all mutant proteins was normal. Thermotolerance of cells expressing mutant proteins was variably resistant to guanidine, and the degree of thermotolerance did not correlate with [ PSI ⁺ ] stability. We thus show that guanidine cures yeast prions by inactivating Hsp104 and identify a highly conserved Hsp104 residue that is critical for yeast prion propagation. Our data suggest that Hsp104 activity can be reduced substantially without affecting [ PSI ⁺ ] stability, and that Hsp104 interacts differently with prion aggregates than with aggregates of thermally denatured protein.

The Saccharomyces cerevisiae [PSI⁺] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI⁺] propagation is impaired by alterations that enhance Ssa1p9s substrate binding. This impairment is overcome by second-site mutations in Ssa1p9s conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI⁺] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI⁺] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI⁺] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI⁺] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI⁺] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.