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.

Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14

“…However, oscillation, chemotropic interactions, and fusion were never observed in the Dham-5; his-3:: ham-5 1-523 -gfp strains. Nuclear localization was also observed for HAM-5 1-351 -GFP that contained the seven WD40 repeats ( Figure 1E, WD40) (Jonkers et al 2014), but no cytoplasmic puncta were observed. Thus, the domain required for nuclear exclusion of HAM-5 is situated between amino acids 523 and 777 and sequences important for localization of HAM-5 to puncta are between amino acids 351 and 523.…”

“…In the filamentous ascomycete fungus, Neurospora crassa, genetically identical germinated asexual spores (germlings) undergo fusion to form a syncytium that is an interconnected network of fused cells Leeder et al 2013;Herzog et al 2015). An important pathway required for both germling and hyphal fusion is a conserved mitogen-activated protein kinase (MAPK) cascade that includes the MAPK kinase kinase NRC-1, the MAPK kinase MEK-2, the MAPK MAK-2, and the scaffold protein, HAM-5 (Pandey et al 2004;Dettmann et al 2012Dettmann et al , 2014Jonkers et al 2014). During germling fusion, chemotrophic growth is associated with oscillation of the MAK-2/MEK-2/NRC-1/HAM-5 complex and the regulatory adaptor subunit STE-50 to the fusion tips of germlings, conidial anastomosis tubes (CATs), and to the tips of hyphae undergoing chemotropic interactions prior to cell fusion (fusion hyphae) (Fleissner et al 2009;Dettmann et al 2012Dettmann et al , 2014Jonkers et al 2014 signaling complex is perfectly out of phase with a second protein, called SOFT, which is also essential for chemotropic interactions and cell fusion (Fleissner et al 2005).…”

“…During germling fusion, chemotrophic growth is associated with oscillation of the MAK-2/MEK-2/NRC-1/HAM-5 complex and the regulatory adaptor subunit STE-50 to the fusion tips of germlings, conidial anastomosis tubes (CATs), and to the tips of hyphae undergoing chemotropic interactions prior to cell fusion (fusion hyphae) (Fleissner et al 2009;Dettmann et al 2012Dettmann et al , 2014Jonkers et al 2014 signaling complex is perfectly out of phase with a second protein, called SOFT, which is also essential for chemotropic interactions and cell fusion (Fleissner et al 2005). Switching between the MAK-2 complex and SOFT at the tips of cells undergoing chemotropic interactions occurs every 4-5 min (Fleissner et al 2009;Jonkers et al 2014). Phosphorylation and subsequent dephosphorylation of HAM-5 has been hypothesized to regulate the assembly and disassembly of the MAK-2 complex at CATs and hyphal fusion tips (Jonkers et al 2014).…”

“…The ham-5 1-351 -gfp construct also failed to complement the growth and fusion defects of the Dham-5 mutant. HAM-5 lacking the WD40 domain (DWD40, Figure 1A) is also nonfunctional and is unstable in germlings and hyphae (Jonkers et al 2014), but punctate localization was detected in conidia ( Figure 1E bottom left). These data indicate that the C terminus of HAM-5 (amino acids 351-1686) is required for function, for nuclear exclusion, and/or for localization to puncta.…”

“…7) strains formed protoperithecia and produced viable ascospores in crosses with wild type. Thus, the functional domains required for germling fusion were also required for sexual development, suggesting similar roles for HAM-5 in these two distinct processes.Previous biochemical approaches showed that the binding of HAM-5 to MAK-2 occurs through the WD40 domain and that binding to MEK-2 occurs at the region C-terminal to the WD40 domain (Jonkers et al 2014). To further dissect the region of interaction between MEK-2 and HAM-5, heterokaryotic strains were constructed bearing ham-5-gfp or the ham-5-gfp truncation alleles with mek-2-mCherry.…”