Kch1 Family Proteins Mediate Essential Responses to Endoplasmic Reticulum Stresses in the Yeasts Saccharomyces cerevisiae and Candida albicans

Stefan, Christopher P.; Cunningham, Kyle W.

doi:10.1074/jbc.m113.508705

Cited by 15 publications

(10 citation statements)

References 70 publications

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“…Activation of Ca 2+ channels, calmodulin, calcineurin and other factors is necessary for the long-term survival of cells undergoing ER stress, and during this process the HACS components, CchA and MidA, are required for Ca 2+ influx from the extracellular environment [ 41 , 50 , 51 ]. To verify whether AkrA is involved in the calcium influx response during ER stress, we measured the influence of the ER-stress agents, tunicamycin (TM) and dithiothreitol (DTT) on [Ca 2+ ] c .…”

Section: Resultsmentioning

confidence: 99%

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Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus

et al. 2016

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Finely tuned changes in cytosolic free calcium ([Ca2+]c) mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS). The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs), a putative proton V-type proton ATPase (Vma5 homolog) and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress.

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Section: Resultsmentioning

confidence: 99%

“…Previous studies have verified that exposure of fungi to ER or plasma membrane stress stimulates store-operated calcium influx through the HACS to promote fungal cell survival [ 13 , 14 , 41 , 50 – 52 ]. Consistent with previous studies, in A .…”

Section: Discussionmentioning

confidence: 99%

Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus

et al. 2016

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“…Trk1p is a component of the Trk1p-Trk2p potassium transport system in yeast, which plays a major role in potassium uptake4445, and was previously shown to be essential for tunicamycin treatment46 and Histatin 5 toxicity47 in C. albicans . Similarly, Trk1p was shown to be required for fungicidal activity by lactoferrin 11, bactenecin 16 and virion-associated protein VPR 12 against C. albicans , pointing to Trk1p as a functional effector of these compounds48.…”

Section: Discussionmentioning

confidence: 99%

The antifungal plant defensin AtPDF2.3 from Arabidopsis thaliana blocks potassium channels

Vriens

Peigneur

Coninck

et al. 2016

Sci Rep

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Scorpion toxins that block potassium channels and antimicrobial plant defensins share a common structural CSαβ-motif. These toxins contain a toxin signature (K-C4-X-N) in their amino acid sequence, and based on in silico analysis of 18 plant defensin sequences, we noted the presence of a toxin signature (K-C5-R-G) in the amino acid sequence of the Arabidopsis thaliana defensin AtPDF2.3. We found that recombinant (r)AtPDF2.3 blocks Kv1.2 and Kv1.6 potassium channels, akin to the interaction between scorpion toxins and potassium channels. Moreover, rAtPDF2.3[G36N], a variant with a KCXN toxin signature (K-C5-R-N), is more potent in blocking Kv1.2 and Kv1.6 channels than rAtPDF2.3, whereas rAtPDF2.3[K33A], devoid of the toxin signature, is characterized by reduced Kv channel blocking activity. These findings highlight the importance of the KCXN scorpion toxin signature in the plant defensin sequence for blocking potassium channels. In addition, we found that rAtPDF2.3 inhibits the growth of Saccharomyces cerevisiae and that pathways regulating potassium transport and/or homeostasis confer tolerance of this yeast to rAtPDF2.3, indicating a role for potassium homeostasis in the fungal defence response towards rAtPDF2.3. Nevertheless, no differences in antifungal potency were observed between the rAtPDF2.3 variants, suggesting that antifungal activity and Kv channel inhibitory function are not linked.

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“…The activity of Kch1 and Kch2 may set up a voltage differential across the plasma membrane, thus activating the HACS complex. Kch1 is conserved in C. albicans ( orf19.6563 ) ( Stefan and Cunningham 2013 ), and in C. glabrata ( CAGL0D06050g ) ( Inglis et al . 2012 ).…”

Section: Discussionmentioning

confidence: 99%

Identification of Genes inCandida glabrataConferring Altered Responses to Caspofungin, a Cell Wall Synthesis Inhibitor

Rosenwald

Arora

Ferrandino

et al. 2016

G3 Genes|Genomes|Genetics

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Candida glabrata is an important human fungal pathogen whose incidence continues to rise. Because many clinical isolates are resistant to azole drugs, the drugs of choice to treat such infections are members of the echinocandin family, although there are increasing reports of resistance to these drugs as well. In efforts to better understand the genetic changes that lead to altered responses to echinocandins, we screened a transposon-insertion library of mutants for strains to identify genes that are important for cellular responses to caspofungin, a member of this drug family. We identified 16 genes that, when disrupted, caused increased tolerance, and 48 genes that, when disrupted, caused increased sensitivity compared to the wild-type parental strain. Four of the genes identified as causing sensitivity are orthologs of Saccharomyces cerevisiae genes encoding proteins important for the cell wall integrity (CWI) pathway. In addition, several other genes are orthologs of the high affinity Ca2+ uptake system (HACS) complex genes. We analyzed disruption mutants representing all 64 genes under 33 different conditions, including the presence of cell wall disrupting agents and other drugs, a variety of salts, increased temperature, and altered pH. Further, we generated knockout mutants in different genes within the CWI pathway and the HACS complex, and found that they too exhibited phenotypes consistent with defects in cell wall construction. Our results indicate that small molecules that inhibit the CWI pathway, or that the HACS complex, may be an important means of increasing the efficacy of caspofungin.

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Kch1 Family Proteins Mediate Essential Responses to Endoplasmic Reticulum Stresses in the Yeasts Saccharomyces cerevisiae and Candida albicans

Cited by 15 publications

References 70 publications

Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus

Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus

The antifungal plant defensin AtPDF2.3 from Arabidopsis thaliana blocks potassium channels

Identification of Genes inCandida glabrataConferring Altered Responses to Caspofungin, a Cell Wall Synthesis Inhibitor

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