Abstract
The vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that play vital roles in eukaryotic cells. Insect V-ATPases are required in nearly all epithelial tissues to regulate a multiplicity of processes including receptor-mediated endocytosis, protein degradation, fluid secretion, and neurotransmission. Composed of fourteen different subunits, several V-ATPase subunits exist in distinct isoforms to perform cell type specific functions. The 100 kD a subunit (Vha100) of V-ATPases are encoded by a family of five genes in Drosophila, but their assignments are not fully understood. Here we report an experimental survey of the Vha100 gene family during Drosophila wing development. A combination of CRISPR-Cas9 mutagenesis, somatic clonal analysis and in vivo RNAi assays is used to characterize the requirement of Vha100 isoforms, and mutants of Vha100-2, Vha100-3, Vha100-4, and Vha100-5 genes were generated. We show that Vha100-3 and Vha100-5 are dispensable for fly development, while Vha100-1 is not critically required in the wing. As for the other two isoforms, we find that Vha100-2 regulates wing cuticle maturation, while Vha100-4 is the single isoform involved in developmental patterning. More specifically, Vha100-4 is required for proper activation of the Wingless signaling pathway during fly wing development. Interestingly, we also find a specific genetic interaction between Vha100-1 and Vha100-4 during wing development. Our results revealed the distinct roles of Vha100 isoforms during insect wing development, providing a rationale for understanding the diverse roles of V-ATPases.
Highlights
The vacuolar ATPases (V-ATPases) are ubiquitous proton pumps which play important roles in eukaryotic cells (Forgac, 2007)
The a subunit of the V-ATPase is encoded by five genes in Drosophila, which are spread at different locations throughout the genome (Supplementary Figures S1A–D)
A number of residues of the a subunit have been experimentally demonstrated to be important for the activity or assembly of V-ATPase complex in yeast (Leng et al, 1996, 1998), which were later found to be conserved in mouse orthologs (Nishi and Forgac, 2000; Toyomura et al, 2000)
Summary
The vacuolar ATPases (V-ATPases) are ubiquitous proton pumps which play important roles in eukaryotic cells (Forgac, 2007). V-ATPases transport proton into intracellular compartments, and are crucial for pH homeostasis in organelles such as endosomes, secretory vesicles, synaptic vesicles, and lysosomes (Hinton et al, 2009). The V-ATPases are generally required for a broad spectrum of cellular processes, including endosomal trafficking, lysosomal degradation, and exocytosis (Forgac, 2007). Insect V-ATPases are expressed in most epithelial tissues and are well-known for their roles in physiological activities such as secretion of K + and. Recent studies have illuminated the importance of V-ATPases for insect development. In Drosophila melanogaster, mutations that dampen the V-ATPases activity are reported to disrupt the formation of eye, wing and egg chambers (Yan et al, 2009; Buechling et al, 2010; Hermle et al, 2010; Vaccari et al, 2010). Further studies demonstrated that V-ATPases are involved in regulation of cell proliferation, cell fate determination and tissue patterning through modification of key developmental signaling pathways (Kobia et al, 2013; Gleixner et al, 2014; Portela et al, 2018; Ren et al, 2018)
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