Abstract
The plasma membrane is implicated in a variety of functions, whose coordination necessitates highly dynamic organization of its constituents into domains of distinct protein and lipid composition. Eisosomes, at least partially, mediate this lateral plasma membrane compartmentalization. In this work, we show that the Nce102 homologue of Aspergillus nidulans colocalizes with eisosomes and plays a crucial role in density/number of PilA/SurG foci in the head of germlings. In addition we demonstrate that AnNce102 and PilA negatively regulate sphingolipid biosynthesis, since their deletions partially suppress the thermosensitivity of basA mutant encoding sphingolipid C4-hydroxylase and the growth defects observed upon treatment with inhibitors of sphingolipid biosynthesis, myriocin and Aureobasidin A. Moreover, we show that YpkA repression mimics genetic or pharmacological depletion of sphingolipids, conditions that induce the production of Reactive Oxygen Species (ROS), and can be partially overcome by deletion of pilA and/or annce102 at high temperatures. Consistent with these findings, pilAΔ and annce102Δ also show differential sensitivity to various oxidative agents, while AnNce102 overexpression can bypass sphingolipid depletion regarding the PilA/SurG foci number and organization, also leading to the mislocalization of PilA to septa.
Highlights
Pil[1] and Lsp[1] by the Pkh1/2 kinase and the levels of sphingolipid Long-Chain Bases (LCBs)[12,13,14]
We studied cells treated with myriocin and Aureobasidin A, specific inhibitors of sphingolipid biosynthesis[25,28], as well as cells genetically blocked for sphingolipid biosynthesis. 20% of myriocin and 36% of AbA treated cells displayed increased Reactive Oxygen Species (ROS) levels, visualized by the ROS indicator dye 2′, 7′ -dichlorofluorescin diacetate (DCF) (Fig. 7A)
In order to test whether the above differences in growth and ROS accumulation are caused by the regulation of sphingolipid biosynthesis, we introduced the pilAΔ and/or annce102Δ mutations in a basA1 strain
Summary
Pil[1] and Lsp[1] by the Pkh1/2 kinase (homologues of mammalian 3-phosphoinositide-dependent kinase) and the levels of sphingolipid Long-Chain Bases (LCBs)[12,13,14]. Ypk[1] phosphorylates and inactivates the endoplasmic reticulum (ER)-localized proteins, Orm[1] and Orm[217,19,20]. It stimulates the function of the ceramide synthase complex, by increasing the rate of the formation of ceramides and preventing hyper-accumulation of LCBs/LCBPs, avoiding inadvertent induction of autophagy under sufficient conditions[21]. In the model filamentous fungus Aspergillus nidulans, PilA, PilB and SurG, homologues of the S. cerevisiae eisosome proteins Pil1/Lsp[1] and Sur[7], are assembled and form tightly packed structures[22]. In addition we show that the main organizers of eisosomes, the PilA and AnNce[102] proteins, negatively regulate sphingolipid biosynthesis
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