Miwha Chang

Identification of high-affinity copper transporters in Aspergillus fumigatus

We investigated the copper metabolism of Aspergillus fumigatus, which has not been characterized well. We cloned the putative copper transporters ctrA2 and ctrC from A. fumigatus and investigated the functions of these transporters in copper metabolism. Four putative copper transporters were identified in the A. fumigatus genome; ctrA2 and ctrC complemented CTR1 functionally and localized to the plasma membrane in Saccharomyces cerevisiae. ctrA2 and ctrC single-deletion mutants and a double-deletion mutant of ctrA2 and ctrC were constructed in A. fumigatus. The ctrA2 and ctrC double-deletion mutant exhibited a growth defect on Aspergillus minimal medium (AMM) supplemented with bathocuproine disulfonic acid (BCS) and was sensitive to H2O2. Furthermore, the deletion of ctrA2 and ctrC reduced superoxide dismutase (SOD) activity, laccase activity, and intracellular copper contents. The activities of the ctrA2 and ctrC genes were up-regulated by BCS treatment. In addition, the deletion of ctrA2 up-regulated ctrC and vice versa. ctrA2 and ctrC were localized to the A. fumigatus plasma membrane. Although ctrA2 and ctrC failed to affect the mouse survival rate, these genes affected conidial killing activity. Taken together, these results indicate that ctrA2 and ctrC may function as membrane transporters and that the involvement of these genes in pathogenicity merits further investigation.

Cadmium regulates copper homoeostasis by inhibiting the activity of Mac1, a transcriptional activator of the copper regulon, in Saccharomyces cerevisiae.

Cadmium is a toxic metal and the mechanism of its toxicity has been studied in various model systems from bacteria to mammals. We employed Saccharomyces cerevisiae as a model system to study cadmium toxicity at the molecular level because it has been used to identify the molecular mechanisms of toxicity found in higher organisms. cDNA microarray and Northern blot analyses revealed that cadmium salts inhibited the expression of genes related to copper metabolism. Western blotting, Northern blotting and chromatin immunoprecipitation experiments indicated that CTR1 expression was inhibited at the transcriptional level through direct inhibition of the Mac1 transcriptional activator. The decreased expression of CTR1 results in cellular copper deficiency and inhibition of Fet3 activity, which eventually impairs iron uptake. In this way, cadmium exhibits a negative effect on both iron and copper homoeostasis.

A novel function of Aft1 in regulating ferrioxamine B uptake: Aft1 modulates Arn3 ubiquitination in Saccharomyces cerevisiae

Aft1 is a transcriptional activator in Saccharomyces cerevisiae that responds to iron availability and regulates the expression of genes in the iron regulon, such as FET3, FTR1 and the ARN family. Using a two-hybrid screen, we found that Aft1 physically interacts with the FOB (ferrioxamine B) transporter Arn3. This interaction modulates the ability of Arn3 to take up FOB. The interaction between Arn3 and Aft1 was confirmed by beta-galactosidase, co-immunoprecipitation and SPR (surface plasmon resonance) assays. Truncated Aft1 had a stronger interaction with Arn3 and caused a higher FOB-uptake activity than full-length Aft1. Interestingly, only full-length Aft1 induced the correct localization of Arn3 in response to FOB. Furthermore, we found Aft1 affected Arn3 ubiquitination. These results suggest that Aft1 interacts with Arn3 and may regulate the ubiquitination of Arn3 in the cytosolic compartment.

The expression of PHO92 is regulated by Gcr1, and Pho92 is involved in glucose metabolism in Saccharomyces cerevisiae

Ydr374c (Pho92) contains a YTH domain in its C-terminal region and is a human YTHDF2 homologue. Previously, we reported that Pho92 regulates phosphate metabolism by regulating PHO4 mRNA stability. In this study, we found that growth of the ∆pho92 strain on SG media was slower than that of the wild type and that PHO92 expression was up-regulated by non-fermentable carbon sources, such as ethanol and glycerol, but not by fermentable carbon sources. Furthermore, two conserved Gcr1-binding regions were identified in the upstream, untranslated region of PHO92. Gcr1 is an important factor involved in the coordinated regulation of glycolytic gene expression. Mutation of two Gcr1-binding sites of the PHO92 upstream region resulted in a growth defect on SD media. Finally, mutagenesis of the Gcr1-binding sites of the PHO92 upstream region and deletion of GCR1 resulted in up-regulation of PHO92, and this resulted from inhibition of PHO4 mRNA degradation. Based on these results, we suggest that Gcr1 regulates the expression of PHO92, and Pho92 is involved in glucose metabolism.

Cd2+ binds to Atx1 and affects the physical interaction between Atx1 and Ccc2 in Saccharomyces cerevisiae

The ATX1 deletion strain of Saccharomyces cerevisiae is more resistant to Cd2+ than the wild-type. To investigate the function of Atx1 in Cd2+ toxicity, we used a metal-binding assay to study the interaction between Atx1 and Cd2+ in vitro. Using circular dichroism and two-hybrid analyses, we found that Atx1 can bind Cd2+ specifically and that Cd2+ binding to Atx1 affects the physical interaction between Atx1 and Ccc2. These results imply that Atx1 delivers Cd2+ to Ccc2 and that this delivery is, at least in part, responsible for Cd2+ toxicity in S. cerevisiae.

Rck1 up-regulates Hog1 activity by down-regulating Slt2 activity in Saccharomyces cerevisiae.

We previously reported that the over-expression of KDX1 up-regulates RCK1 gene expression. To further understand the function of Rck1, microarray analysis was performed using a RCK1 over-expressing strain. Based on microarray and Northern blot analyses, we determined that the expression of KDX1 was down-regulated when RCK1 was over-expressed. Furthermore, we determined that phosphorylated forms of Slt2 and Mkk2 were down-regulated by the over-expression of RCK1. Ptp2, a phosphatase that is regulated by the Slt2 MAP kinase pathway, was down-regulated by the over-expression of RCK1. Ptp2 is a negative regulator of Hog1; thus, the phosphorylated form of Hog1 was up-regulated by RCK1 over-expression. A point mutation of lysine 152 to arginine resulted in a failure to up-regulate Hog1 and the subsequent down-regulation of CTT1, which is a Hog1 pathway target gene. Furthermore, using microarray and Northern blot analyses, we determined that genes that are regulated by Msn2/Msn4 were up-regulated by Rck1 and that this was the result of Hog1 activation by RCK1 over-expression. Together, our results suggest that Rck1 inhibits Slt2 MAP kinase pathway activity and then Ptp2, which subsequently activates Hog1.

Cadmium inhibits the protein degradation of Sml1 by inhibiting the phosphorylation of Sml1 in Saccharomyces cerevisiae

Cadmium is a toxic metal, and the mechanism of cadmium toxicity in living organisms has been well studied. Here, we used Saccharomyces cerevisiae as a model system to examine the detailed molecular mechanism of cell growth defects caused by cadmium. Using a plate assay of a yeast deletion mutant collection, we found that deletion of SML1, which encodes an inhibitor of Rnr1, resulted in cadmium resistance. Sml1 protein levels increased when cells were treated with cadmium, even though the mRNA levels of SML1 remained unchanged. Using northern and western blot analyses, we found that cadmium inhibited Sml1 degradation by inhibiting Sml1 phosphorylation. Sml1 protein levels increased when cells were treated with cadmium due to disruption of the dependent protein degradation pathway. Furthermore, cadmium promoted cell cycle progression into the G2 phase. The same result was obtained using cells in which SML1 was overexpressed. Deletion of SML1 delayed cell cycle progression. These results are consistent with Sml1 accumulation and with growth defects caused by cadmium stress. Interestingly, although cadmium treatment led to increase Sml1 levels, intracellular dNTP levels also increased because of Rnr3 upregulation due to cadmium stress. Taken together, these results suggest that cadmium specifically affects the phosphorylation of Sml1 and that Sml1 accumulates in cells.

Rck1 up-regulates pseudohyphal growth by activating the Ras2 and MAP kinase pathways independently in Saccharomyces cerevisiae.

Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD+1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae.