Abstract
The NADPH oxidases (NOX) catalyze the production of superoxide by transferring electrons from NADPH to O2, in a regulated manner. In Neurospora crassa NOX-1 is required for normal growth of hyphae, development of aerial mycelium and asexual spores, and it is essential for sexual differentiation and cell-cell fusion. Determining the subcellular localization of NOX-1 is a critical step in understanding the mechanisms by which this enzyme can regulate all these different processes. Using fully functional versions of NOX-1 tagged with mCherry, we show that in growing hyphae NOX-1 shows only a minor association with the endoplasmic reticulum (ER) markers Ca2+-ATPase NCA-1 and an ER lumen-targeted GFP. Likewise, NOX-1 shows minor co-localization with early endosomes labeled with YPT-52, a GTPase of the Rab5 family. In contrast, NOX-1 shows extensive co-localization with two independent markers of the entire vacuolar system; the vacuolar ATPase subunit VMA-1 and the fluorescent molecule carboxy-DFFDA. In addition, part of NOX-1 was detected at the plasma membrane. The NOX-1 regulatory subunit NOR-1 displays a very different pattern of localization, showing a fine granular distribution along the entire hypha and some accumulation at the hyphal tip. In older hyphal regions, germinating conidia, and conidiophores it forms larger and discrete puncta some of which appear localized at the plasma membrane and septa. Notably, co-localization of NOX-1 and NOR-1 was mainly observed under conidial cell-cell fusion conditions in discrete vesicular structures. NOX functions in fungi have been evaluated mainly in mutants that completely lacked this protein, also eliminating interactions between hyphal growth regulatory proteins NOR-1, the GTPase RAC-1 and the scaffold protein BEM-1. To dissect NOX-1 roles as scaffold and as ROS-producing enzyme, we analyzed the function of NOX-1::mCherry proteins carrying proline 382 by histidine (P382H) or cysteine 524 by arginine (C524R) substitutions, predicted to only affect NADPH-binding. Without notably affecting NOX-1 localization or protein levels, each of these substitutions resulted in lack of function phenotypes, indicating that NOX-1 multiple functions are all dependent on its oxidase activity. Our results open new interpretations to possible NOX functions, as components of the fungal vacuolar system and the plasma membrane, as well as to new vacuolar functions.
Highlights
Many years ago we proposed reactive oxygen species (ROS) as signaling molecules and regulators of cell differentiation (Hansberg and Aguirre, 1990; Aguirre et al, 2005)
We reported the presence of three different NADPH oxidase (NOX) subfamilies in fungi and the Amoebozoa, and demonstrated that NOX enzymes were essential for ROS production and sexual development in the Ascomycetes Aspergillus nidulans (LaraOrtiz et al, 2003; Aguirre et al, 2005), Neurospora crassa (CanoDominguez et al, 2008), and Sordaria macrospora (Dirschnabel et al, 2014)
As reported before (CanoDominguez et al, 2008), ∆nox-1 mutants showed a severe decrease in aerial mycelium growth and conidiation, reduced radial growth and were unable to develop perithecia, with these phenotypes being somewhat enhanced by the presence of his-3 mutation (Figures S2A–E)
Summary
Many years ago we proposed reactive oxygen species (ROS) as signaling molecules and regulators of cell differentiation (Hansberg and Aguirre, 1990; Aguirre et al, 2005). More recently the functions of other NOX enzymes, originally considered as ferric reductases, have been reported in Saccharomyces cerevisiae (Rinnerthaler et al, 2012) and Candida albicans (Rossi et al, 2017), while bioinformatic analysis and in vitro experiments suggest the presence of NOX enzymes in prokaryotes (Hajjar et al, 2017). Such ubiquity of NOX enzymes supports the idea that the controlled production of ROS as signaling molecules appeared early in life evolution as an important mechanism to regulate cell physiology and differentiation
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