Analysis and Inactivation of vha55, the Gene Encoding the Vacuolar ATPase B-subunit in Drosophila melanogaster Reveals a Larval Lethal Phenotype

Abstract

Vacuolar ATPases play major roles in endomembrane and plasma membrane proton transport in eukaryotes.A Drosophila melanogaster cDNA encoding vha55, the 55-kDa vacuolar ATPase (V-ATPase) regulatory B-subunit, was characterized and mapped to 87C2-4 on chromosome 3R. A fly line was identified that carried a single lethal P-element insertion within the coding portion of gene, and its LacZ reporter gene revealed elevated expression in Malpighian tubules, rectum, antennal palps, and oviduct, regions where V-ATPases are believed to play a plasma membrane, rather than an endomembrane, role. The P-element vha55 insertion was shown to be allelic to a known lethal complementation group l(3)SzA ‫؍(‬ l(3)87Ca) at 87C, for which many alleles have been described previously. Deletions of the locus have been shown to be larval lethal, whereas point mutations show a range of phenotypes from subvital to embryonic lethal, implying that severe alleles confer a partial dominant negative phenotype. The P-element null allele of vha55 was shown also to suppress ectopic sex combs in Polycomb males, suggesting that transcriptional silencing may be modulated by genes other than those with known homeotic or DNA binding functions. The vacuolar ATPase1 is a multisubunit complex, related to the F 1 /F 0 ATPase (1-3). The transmembrane protonophore is made of six copies of a 16-kDa proteolipid, linked by further subunits to a catalytic headgroup comprising three copies each of a 67-kDa A-subunit and a 57-kDa B-subunit. Traditionally, the A-subunit is described as catalytic, whereas the B-subunit is considered regulatory, although in reality the active sites for nucleotide binding and proton flux may lie in the interfaces between neighboring A-and B-subunits (4). Although there is known to be very high conservation within the V-ATPase family, the 57-kDa subunit is interesting as several transcripts are known, some of which are tissue-specific (5, 6). It has been argued further that choice of B-subunit transcript may affect the overall subunit composition of the holoenzyme by influencing the choice of other subunits during assembly of the V 1 headgroup (6), and the B-subunit is phosphorylated by a component of AP-2, the clathrin assembly complex (7), suggesting a role in control of vesicle trafficking.V-ATPases, although originally defined as endosomal, are now known to energize plasma membrane transport in a variety of tissues, such as kidney, osteoclasts, and frog skin (8); and loss of kidney V-ATPase function in autoimmune disease is clinically significant (9). In an invertebrate model, a painstaking biochemical purification of particles on the goblet cell apical membranes of lepidopteran midgut (10) showed that the invertebrate K ϩ pump (11) was in fact a V-ATPase (12), driving a K ϩ /H ϩ antiporter (13) to produce a remarkably potent transport system (14, 15). Monospecific antibodies against the lepidopteran plasma membrane V-ATPase were used to demonstrate plasma membrane V-ATPases immunocytochemically in salivary glands, Malpighian (renal)...

The vacuolar H+-ATPase: a universal proton pump of eukaryotes

“…The library has been selected on the basis that P-element insertion causes lethality in homozygous flies. To date, five such flies have been identified with insertions in loci encoding VATPase subunits, including four from V " (subunits A, B, E and F) and one from V o (subunit c) [63,64]. The lethality occurs in embryonic or larval stages, emphasizing the importance of the VATPase in higher eukaryotes.…”

“…The ha55 gene in Drosophila encodes subunit B, and P-element inactivation of this gene is embryonic lethal [63]. An unexpected finding was that the gene localizes to a previously identified locus that suppresses polycomb.…”

Proteomic changes in response to crystal formation inDrosophilaMalpighian tubules

“…16 V-ATPase plays a central role in many aspects of cellular function in the kidney as well in other organs. 17,18 These range from proton secretion in order to acidify the extracellular milieu, to intracellular acidification of vesicles and regulation of a variety of processes that range from lysosomal degradation, ligand receptor dissociation and intracellular trafficking. 19 The holoenzyme is comprised of many different subunits, each having different functions that are involved not only in the activity of the enzyme but also to its intracellular regulation and targeting.…”

Characterization and Localization of the Vacuolar-Type ATPase in the Midgut Cells of Silkworm (Bombyx mori)

“…Traditionally, the A subunit is described as catalytic, whereas the B subunit is considered as regulatory, although in reality the active sites for nucleotide binding and proton fl ux may lie in the interfaces between the neighbouring A and B subunits (Liu et al, 1996). In the Drosophila melanogaster VATPase B subunit, the PPVNVLPSLS motif is essential for the ATPase function (Davies et al, 1996). This motif was also found in the B subunit of B. mori and other insects.…”

“…According to the NCBI database, the SAAGLPHN motif is a degenerated motif, based on similarity to F1 ATPases, and the ILTD motif coordinates the Mg 2+ transport. The V-ATPase plays major roles in endomembrane and plasma membrane proton transport in eukaryotes; an inactivated V-ATPase B subunit caused a larval lethal phenotype in Drosophila melanogaster (Davies et al, 1996).…”