Changes in phosphorylation states of the NDR kinase COT1 alter lipid composition in Neurospora crassa

Carmit Ziv carmit.ziv@agri.gov.il ¹ Liran Aharoni Kats ² Valeria Costantino ³ Alfonso Mangoni ³ Roberta Teta ³ Oded Yarden ²

¹Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel
²Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel
³Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Napoli, Italy

The Neurospora crassa NDR kinase COT1 is involved in the regulation of hyphal elongation and branching. COT1 dysfunction results in ultrastructural irregularities, including thickened cell walls, the presence of many cytoplasmic vacuoles, some with vesicle-like inclusions, as well as abnormally shaped mitochondria and nuclei. COT1 has been shown to be associated with the cytoplasmic membrane. This localization, along with COT1 phosphorylation, are required for its proper Ser/Thr protein kinase activity.

In order to investigate the changes that occur in the cell membrane composition, which are dependent on COT1 activity, we applied a liquid chromatography-mass spectrometry (LC-MS)-based lipidomics approach to a set of cot-1 strains, mutated in three conserved phosphorylation sites of the kinase, mimicking non-phosphorylated or constitutively phosphorylated residues of the protein. A mutation in COT1 Thr589, which markedly affects COT1 activity and hyphal morphology, was accompanied with a significantly altered lipid composition that was characterized by an increase in unsaturated glycerolipids, namely diacylglyceols (DAG), triacylglyceols (TAG), phosphatidylethanolamines (PE) and phosphatidylcholines (PC). Furthermore, the phosphomimetic state of COT1 Thr589 strongly affected the saturation profile of the glycosylceramides (GSL) fatty acid components, as well as methylation of their long chain bases (LCBs). GSLs are key components of the plasma membrane and their presence is linked with membrane fluidity and stability, as well as cell signaling and protein sorting. The involvement of GSLs in COT1 dependent morphogenesis was further demonstrated by differential sensitivity of the cot-1 strains to the serine-palmitoyl transferase inhibitor Myriocin and the UDP-glucose:Cer glucosyltransferase inhibitor PDMP.

Only minor changes in transcription of genes involved in GSL biosynthesis were observed in a cot-1 background, suggesting that most of the regulation of COT1-mediated GSL biosynthesis is post transcriptional. The data presented here provide first evidence for the involvement of an NDR kinase in fungal lipid metabolism.