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

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 NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this 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 MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization.more » However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.« less
 [1] ;  [1] ;  [2] ;  [2] ;  [3] ;  [1] ;  [2] ;  [2] ;  [1] ;  [4]
  1. Univ. of California, Berkeley, CA (United States)
  2. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  3. Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Genentech, South San Francisco, CA (United States)
  4. Duke Univ. Medical Center, Durham, NC (United States)
Publication Date:
OSTI Identifier:
Report Number(s):
Journal ID: ISSN 1553-7404; 48135; 400412000
Grant/Contract Number:
AC05-76RL01830; AC02-05CH11231
Accepted Manuscript
Journal Name:
PLoS Genetics
Additional Journal Information:
Journal Volume: 10; Journal Issue: 11; Journal ID: ISSN 1553-7404
Public Library of Science
Research Org:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
60 APPLIED LIFE SCIENCES; Environmental Molecular Sciences Laboratory; cell fusion; phosphorylation; fluorescence imaging; genetic oscillators; Neurospora crassa; immunoprecipitation; MAPK signaling cascades; bright field imaging