title: The essential phosphoinositide kinase MSS-4 is required for polar hyphal morphogenesis, localizing to sites of growth and cell fusion in Neurospora crassa.
abstract: Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P(2). In N. crassa hyphae, PtdIns(4,5)P(2) localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P(2) that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.
authors.affiliation: Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University Göttingen, Göttingen, Germany.
authors.forename: Anette
authors.initials: A
authors.lastname: Mähs
authors.forename: Till
authors.initials: T
authors.lastname: Ischebeck
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Heilig
authors.forename: Irene
authors.initials: I
authors.lastname: Stenzel
authors.forename: Franziska
authors.initials: F
authors.lastname: Hempel
authors.forename: Stephan
authors.initials: S
authors.lastname: Seiler
authors.forename: Ingo
authors.initials: I
authors.lastname: Heilmann
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism*; D000483:Alleles; D002459:Cell Fusion; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D025301:Hyphae / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D008957:Models, Genetic; D016296:Mutagenesis; D009154:Mutation; D009492:Neurospora crassa / Q000201:enzymology*; D016366:Open Reading Frames; D010641:Phenotype; D010716:Phosphatidylinositols / Q000737:chemistry; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011058:Pollen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019870:1-Phosphatidylinositol 4-Kinase; C581878:MSS4 protein, Neurospora crassa; C489795:MSS4 protein, S cerevisiae