title: HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa.
abstract: Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication.
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States of America.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Jonkers
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States of America.
authors.forename: Abigail C
authors.initials: AC
authors.lastname: Leeder
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington, United States of America.
authors.forename: Charles
authors.initials: C
authors.lastname: Ansong
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington, United States of America.
authors.forename: Yuexi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington, United States of America.
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.affiliation: Energy Biosciences Institute, University of California, Berkeley, Berkeley, California, United States of America.
authors.forename: Trevor L
authors.initials: TL
authors.lastname: Starr
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington, United States of America.
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington, United States of America.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States of America; Energy Biosciences Institute, University of California, Berkeley, Berkeley, California, United States of America.
authors.forename: N Louise
authors.initials: NL
authors.lastname: Glass
mesh_terms: D002459:Cell Fusion; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D000071677:Histidine Kinase; D025301:Hyphae / Q000235:genetics / Q000254:growth & development; D048370:MAP Kinase Kinase 2 / Q000235:genetics* / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics*; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D013172:Spores, Fungal / Q000235:genetics / Q000254:growth & development
chemical_list: D005656:Fungal Proteins; D008565:Membrane Proteins; D011494:Protein Kinases; C119849:nrc-1 protein, Neurospora crassa; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048370:MAP Kinase Kinase 2; D000071677:Histidine Kinase