A Mini HIP HOP Assay Uncovers a Central Role for Copper and Zinc in the Antifungal Mode of Action of Allicin

Garlic contains the organosulfur compound allicin which exhibits potent antifungal activity. Here we demonstrate the use of a highly simplified yeast chemical genetic screen to characterize its mode of action. By screening 24 validated yeast gene deletion “signature” strains for which hypersensitivity is characteristic for common antifungal modes of action, yeast lacking the high affinity Cu²⁺ transporter Ctr1 was found to be hypersensitive to allicin. Focusing on transition metal related genes identified two more hypersensitive strains lacking the Cu²⁺ and Zn²⁺ transcription factors Mac1 and Zap1. Hypersensitivity in these strains was reversed by the addition of Cu²⁺ and Zn²⁺ ions, respectively. The results suggest the antifungal activity of allicin is mediated through restricted Cu²⁺ and Zn²⁺ uptake or inhibition of Cu²⁺ and Zn²⁺ metalloproteins. As certain antimicrobial modes of action are much more common than others, the approach taken here provides a useful way to identify them early on

ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo

Hsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90-specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP-coupled chaperone cycle for Hsp90-mediated protein folding

Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery

Cellular homeostasis relies on both the chaperoning of proteins and the intracellular degradation system that delivers cytoplasmic constituents to the lysosome, a process known as autophagy. The crosstalk between these processes and their underlying regulatory mechanisms is poorly understood. Here, we show that the molecular chaperone heat shock protein 90 (Hsp90) forms a complex with the autophagy-initiating kinase Atg1 (yeast)/Ulk1 (mammalian), which suppresses its kinase activity. Conversely, environmental cues lead to Atg1/Ulk1-mediated phosphorylation of a conserved serine in the amino domain of Hsp90, inhibiting its ATPase activity and altering the chaperone dynamics. These events impact a conformotypic peptide adjacent to the activation and catalytic loop of Atg1/Ulk1. Finally, Atg1/Ulk1-mediated phosphorylation of Hsp90 leads to dissociation of the Hsp90:Atg1/Ulk1 complex and activation of Atg1/Ulk1, which is essential for initiation of autophagy. Our work indicates a reciprocal regulatory mechanism between the chaperone Hsp90 and the autophagy kinase Atg1/Ulk1 and consequent maintenance of cellular proteostasis.</p