Protein kinase C regulates the expression of cell wall-related genes in RlmA-dependent and independent manners in <i>Aspergillus nidulans</i>

<div><p>A protein kinase C of <i>Aspergillus nidulans</i>, PkcA, is required for cell wall integrity (CWI) and is considered a major component of the regulating pathway. To investigate whether PkcA regulates the transcription of cell wall-related genes, we constructed strains expressing <i>pkcA</i>(R429A) that encodes an activated form of PkcA. The mRNA levels of most chitin synthase genes and an α-glucan synthase gene, <i>agsB</i>, were increased when <i>pkcA</i>(R429A) expression was induced. These mRNA increases were not observed or were only partially observed, in a deletion mutant of <i>rlmA</i>, an ortholog of <i>RLM1</i> that encodes a transcription factor in the CWI pathway in <i>Saccharomyces cerevisiae</i>. In addition, in a <i>pkcA</i> temperature-sensitive mutant under heat stress, the mRNA levels of some chitin synthase genes and <i>agsB</i> did not increase. These results suggest that PkcA is involved in CWI maintenance through the transcriptional regulation of cell wall-related genes.</p></div

Involvement of Protein Kinase C in the Suppression of Apoptosis and in Polarity Establishment in <em>Aspergillus nidulans</em> under Conditions of Heat Stress

<div><p>The <em>pkcA</em> gene, which encodes a protein kinase C (PKC) in the filamentous fungus <em>Aspergillus nidulans</em>, is essential for its viability. However, little is known about its functions. To address this issue, we constructed and characterized temperature-sensitive mutants of <em>pkcA</em>. The conidia of these mutants swelled slightly and exhibited apoptotic phenotypes at 42°C. The apoptotic phenotypes were suppressed by an osmotic stabilizer. Under these conditions, the conidia swelled extensively and did not form germ tubes. Moreover, polarized distribution of F-actin was not observed. We then utilized deletion mutants of <em>bckA</em>, an ortholog of <em>Saccharomyces cerevisiae bck1</em> that encodes a mitogen-activated protein (MAP) kinase kinase kinase and functions downstream of PKC in the cell wall integrity pathway. These mutants exhibited apoptotic phenotypes at 42°C, but they did not show defects in polarity establishment under osmotically stabilized conditions. These results suggest that PkcA plays multiple roles during germination under conditions of heat stress. The first of these roles is the suppression of apoptosis induction, while the other involves polarity establishment. The former depends on the MAP kinase cascade, whereas the latter does not. In addition, repolarization, which was observed after depolarization in the wild-type strain and the <em>bckA</em> deletion mutant under conditions of heat stress, was not observed in the <em>pkcA</em>-ts mutant. This suggests that PkcA also plays role in polarity establishment during hyphal growth independent of the MAP kinase cascade under these conditions.</p> </div

Evaluation of sterol transport from the endoplasmic reticulum to mitochondria using mitochondrially targeted bacterial sterol acyltransferase in <i>Saccharomyces cerevisiae</i>

<div><p>To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a <i>Saccharomyces cerevisiae</i> mutant deleted for <i>ARE1</i> and <i>ARE2</i> encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.</p></div

DNA degradation occurred in the <i>pkcA</i>-ts mutant at 42°C during germination.

<p>A. The viabilities of the conidia of the A1149 strain (Wild-type) and the pkcA-ts-2 mutant (<i>pkcA</i>-ts) incubated in the YG medium for indicated time periods at 42°C. Data are shown as means ± S.E.M, of three independent experiments. B. Conidia from the A1149 strain (Wild-type) and the pkcA-ts-2 mutant (<i>pkcA</i>-ts) were incubated in the YG medium for 5 h at 42°C, and then fixed and stained with DAPI. Bar, 5 µm. C. Conidia from the A1149 strain (Wild-type) and the pkcA-ts-2 mutant (<i>pkcA</i>-ts) were incubated in the YG medium for indicated times at 42°C, and analyzed by flow cytometry. When the wild-type strain was incubated at 42°C, sharp peak was not observed at 2 h of incubation. The cause of this phenomenon is currently unclear.</p

The <i>pkcA</i>-ts mutant exhibited apoptotic phenotypes at 42°C.

<p>A. Conidia from the A1149 strain (Wild-type) and the pkcA-ts-2 mutant (<i>pkcA</i>-ts) were incubated in the YG medium for 4 h at 42°C and treated with H₂DCFDA. Conidia from the A1149 strain pretreated with farnesol were treated with H₂DCFDA and indicated as control. Bar, 10 µm. B. The ratios of cells stained with H₂DCFDA under the conditions of panel A. Data are shown as means ± S.E.M, of three independent experiments. C. Conidia from the A1149 strain (Wild-type) and the pkcA-ts-2 mutant (<i>pkcA</i>-ts) were inoculated in the liquid YG medium on coverslips, incubated for 8 h at 42°C and fixed, labeled with TUNEL and stained with DAPI. Conidia from the A1149 strain pretreated with farnesol were labeled with TUNEL and stained with DAPI, and indicated as control. Bar, 10 µm.</p

The osmotic stabilizer suppresses the DNA degradation of the <i>pkcA</i>-ts mutant at 42°C.

<p>A. Conidia from the A1149 strain (Wild-type) and the pkcA-ts-2 mutant (<i>pkcA</i>-ts) were incubated in the YG medium supplemented with 1.2 M sorbitol for indicated time periods at 42°C, and then analyzed by flow cytometry. B. Conidia from the A1149 strain (Wild-type) and the pkcA-ts-2 mutant (<i>pkcA</i>-ts) were incubated in the YG medium supplemented with 1.2 M sorbitol for 9 h at 42°C, and then fixed and stained with DAPI. Bar, 10 µm.</p

The sensitivity to farnesol of the pkcA-ts-2 mutant.

<p>Aliquots (5 ml) of 10-fold dilutions derived from a starting suspention of 1.0×10⁸ conidia ml⁻¹ of the A1149 strain (wild-type) and the pkcA-ts-2 mutant (<i>pkcA</i>-ts) (A), the A1149/pyroA strain (wild-type) and the <i>bckA</i> deletion mutant (Δ<i>bckA</i>) (B), and the A1149/pyrG-1 strain (wild-type) and the <i>mpkA</i> deletion mutant (Δ<i>mpkA</i>) (C) were spotted on the YG plates supplemented with the 0, 0.1, 0.5 mM farnesol, and the A1149 strain (wild-type) the alcA(p)-pkcA-3 strain (<i>alcA</i>(p)-<i>pkcA</i>) (D) were spotted on the indicated plates supplemented with the 0, 0.1, 0.5 mM. The plates were incubated at the indicated temperature for 48 h.</p

<i>pkcA</i> is required for the repolarization after the depolarization by heat stress.

<p>A. Conidia from the A1149LA-1 strain (Wild-type), the pkcA-tsLA-1 strain (<i>pkcA</i>-ts) and the ΔbckALA-1 strain (Δ<i>bckA</i>) were incubated in the YT medium on coverslips for 16 h (A1149LA-1 and pkcA-tsLA-1) or 24 h (ΔbckALA-1) at 30°C, shifted to 42°C, and observed after indicated time periods. Bar, 5 µm. B. The percentage of hyphae in which Lifeact-EGFP was observed at the tips was measured under the same condition as panel A. Data are shown as means ± S.E.M, of three independent experiments. C. Conidia from the pkcA-tsLA-1 strain were incubated in the YT medium on coverslips for 16 h at 30°C, shifted to 42°C, and observed 60 min after the shift (left panels). They were incubated for 60 min at 30°C after the incubation for 60 min at 42°C and observed (right panels). Bar, 5 µm. D. The percentage of hyphae in which Lifeact-EGFP was observed at the tips was measured when conidia of the pkcA-tsLA-1 strain incubated at 30°C were shifted to 42°C for indicated time periods (empty diamonds). The pkcA-tsLA-1 strain shifted to 30°C after the incubation for 60 min at 42°C was shown by filled rectangles.</p

A model for the functions of PkcA at 42

<p>°<b>C.</b> The functions of PkcA during germination and tip growth suggested in this study are shown in this figure. Hatched circles represent conidia. Involvement of MkkA in the signal transduction pathway for the suppression of apoptosis induction was not investigated in this study. “ ? ” means the possible presence of unknown factors.</p

Lifeact-EGFP can be used as a reporter to detect F-actin in <i>A. nidulans</i>.

<p>A. 10³ conidia from the A1149/pyrG-1 strain (Wild-type) and the A1149LA-1 strain (Lifeact-EGFP) were inoculated under the <i>alcA</i>(p)-repressing condition (YG) and the <i>alcA</i>(p)-inducing condition (YTF), and incubated for 72 h at 37°C. B. Conidia from the A1149LA-1 strain (Lifeact-EGFP) were grown in the YT medium at 37°C for 4 h (isotropic growth) or for 5 h (germ tube formation), or incubated on coverslips submerged in the supplemented MMTF medium for 30 h (hyphal growth and septum formation) or in the supplemented MMG medium for 30 h (<i>alcA</i>(p)-repressing condition). Bar, 5 µm. C. Conidia of the A1149LA-1 strain (Lifeact-EGFP) were inoculated on coverslips and incubated in the MMTF medium for 16 h at 37°C, and treated with Cytochalasin A. Bar, 5 µm.</p