skip to main content

DOE PAGESDOE PAGES

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
Authors:
 [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:
Report Number(s):
PNNL-SA-103955
Journal ID: ISSN 1553-7404; 48135; 400412000
Grant/Contract Number:
AC05-76RL01830; AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
PLoS Genetics
Additional Journal Information:
Journal Volume: 10; Journal Issue: 11; Journal ID: ISSN 1553-7404
Publisher:
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
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; Environmental Molecular Sciences Laboratory; cell fusion; phosphorylation; fluorescence imaging; genetic oscillators; Neurospora crassa; immunoprecipitation; MAPK signaling cascades; bright field imaging
OSTI Identifier:
1166844
Alternate Identifier(s):
OSTI ID: 1407220

Jonkers, Wilfried, Leeder, Abigail C., Ansong, Charles, Wang, Yuexi, Yang, Feng, Starr, Trevor L., Camp, II, David G., Smith, Richard D., Glass, N. Louise, and Heitman, Joseph. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa. United States: N. p., Web. doi:10.1371/journal.pgen.1004783.
Jonkers, Wilfried, Leeder, Abigail C., Ansong, Charles, Wang, Yuexi, Yang, Feng, Starr, Trevor L., Camp, II, David G., Smith, Richard D., Glass, N. Louise, & Heitman, Joseph. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa. United States. doi:10.1371/journal.pgen.1004783.
Jonkers, Wilfried, Leeder, Abigail C., Ansong, Charles, Wang, Yuexi, Yang, Feng, Starr, Trevor L., Camp, II, David G., Smith, Richard D., Glass, N. Louise, and Heitman, Joseph. 2014. "HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa". United States. doi:10.1371/journal.pgen.1004783. https://www.osti.gov/servlets/purl/1166844.
@article{osti_1166844,
title = {HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa},
author = {Jonkers, Wilfried and Leeder, Abigail C. and Ansong, Charles and Wang, Yuexi and Yang, Feng and Starr, Trevor L. and Camp, II, David G. and Smith, Richard D. and Glass, N. Louise and Heitman, Joseph},
abstractNote = {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. 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.},
doi = {10.1371/journal.pgen.1004783},
journal = {PLoS Genetics},
number = 11,
volume = 10,
place = {United States},
year = {2014},
month = {11}
}