Cano-Domínguez Nallely, Bowman Barry, Peraza-Reyes Leonardo, Aguirre Jesús
Departamento de Biología Celular y del Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA, United States.
Front Microbiol. 2019 Aug 14;10:1825. doi: 10.3389/fmicb.2019.01825. eCollection 2019.
The NADPH oxidases (NOX) catalyze the production of superoxide by transferring electrons from NADPH to O, in a regulated manner. In 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 Ca-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.
NADPH氧化酶（NOX）通过将电子从NADPH以一种受调控的方式转移到O₂来催化超氧化物的产生。在[具体真菌名称未给出]中，NOX - 1是菌丝正常生长、气生菌丝体和无性孢子发育所必需的，并且对于有性分化和细胞 - 细胞融合至关重要。确定NOX - 1的亚细胞定位是理解该酶调控所有这些不同过程机制的关键步骤。使用标记有mCherry的功能完全正常的NOX - 1版本，我们发现，在生长的菌丝中，NOX - 1仅与内质网（ER）标记物钙ATP酶NCA - 1和靶向ER腔的绿色荧光蛋白有轻微关联。同样，NOX - 1与用Rab5家族的GTP酶YPT - 52标记的早期内体有轻微共定位。相比之下，NOX - 1与整个液泡系统的两个独立标记物有广泛共定位；液泡ATP酶亚基VMA - 1和荧光分子羧基 - DFFDA。此外，在质膜上检测到部分NOX - 1。NOX - 1调节亚基NOR - 1显示出非常不同的定位模式，沿整个菌丝呈现精细的颗粒分布，并在菌丝尖端有一些积累。在较老的菌丝区域、萌发的分生孢子和分生孢子梗中，它形成更大且离散的斑点，其中一些似乎定位在质膜和隔膜处。值得注意的是，NOX - 1和NOR - 1的共定位主要在分生孢子细胞 - 细胞融合条件下的离散囊泡结构中观察到。真菌中NOX的功能主要在完全缺乏该蛋白的突变体中进行了评估，这也消除了菌丝生长调节蛋白NOR - 1、GTP酶RAC - 1和支架蛋白BEM - 1之间的相互作用。为了剖析NOX - 1作为支架和作为活性氧产生酶的作用，我们分析了携带组氨酸取代脯氨酸382（P382H）或精氨酸取代半胱氨酸524（C524R）的NOX - 1::mCherry蛋白的功能，预计这些取代仅影响NADPH结合。在不显著影响NOX - 1定位或蛋白水平的情况下，这些取代中的每一个都导致了功能缺失表型，表明NOX - 1的多种功能都依赖于其氧化酶活性。我们的结果为NOX作为真菌液泡系统和质膜的组成部分的可能功能以及新的液泡功能开辟了新的解释。
