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PLoS Genet. 2014 Nov 20;10(11):e1004783. doi: 10.1371/journal.pgen.1004783. eCollection 2014 Nov.

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa.

Author information

1
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States of America.
2
Pacific Northwest National Laboratory, Richland, Washington, United States of America.
3
Energy Biosciences Institute, University of California, Berkeley, Berkeley, California, United States of America.
4
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.

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.

PMID:
25412208
PMCID:
PMC4238974
DOI:
10.1371/journal.pgen.1004783
[Indexed for MEDLINE]
Free PMC Article

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