A Novel Insect V-ATPase Subunit M9.7 Is Glycosylated Extensively

Abstract

Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypeptide. Based on N-terminal protein sequencing, we cloned a corresponding cDNA. The deduced hydrophobic protein consisted of 88 amino acids with a molecular mass of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypeptide, using a monoclonal antibody to the 20-kDa polypeptide, confirmed that the correct cDNA had been cloned. The 20-kDa polypeptide is glycosylated, as deduced from lectin staining. Treatment with N-glycosidase A resulted in the appearance of two additional protein bands of 16 and 10 kDa which both were immunoreactive to the 20-kDa polypeptidespecific monoclonal antibody. Thus, extensive N-glycosylation of the novel V o subunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophoresis. M9.7 exhibits some similarities to the yeast protein Vma21p which resides in the endoplasmic reticulum and is required for the assembly of the V o complex. However, as deduced from immunoblots as well as from activities of the V-ATPase and endoplasmic reticulum marker enzymes in different membrane preparations, M9.7 is, in contrast to the yeast polypeptide, a constitutive subunit of the mature plasma membrane V-ATPase of M. sexta. Hϩ V-ATPases are a class of ion transport proteins that couple ATP hydrolysis to the movement of protons across membranes. In endomembranes they function, in concert with chloride channels, as acidifiers of intracellular compartments, whereas in plasma membranes their roles are dependent on the cell type. V-ATPases consist of a peripheral V 1 complex, which is responsible for the hydrolysis of ATP, and a membranebound V o complex which is responsible for the translocation of protons. Although the subunit composition may depend upon the source of the enzyme, at least seven subunits of the V 1 complex, subunits A to G, appear to be universal V-ATPase components (1). By contrast, the subunit composition of the V o complex is less clear. There is no doubt that a 16 -17-kDa proteolipid, the proton "channel," is a major constituent of the V o complex. A membrane-associated subunit in the 40-kDa range and an ϳ100-kDa transmembrane subunit may be two additional essential V o components (1). Recently, a novel 9.2-kDa membrane sector-associated polypeptide was reported from bovine chromaffin granules (2). Its sequence and structure show some similarity to Vma21p, a yeast protein involved in the assembly of the V-ATPase; whether or not it is a constitutive V-ATPase subunit remains an open question.In the larval midgut epithelium of the model insect, Manduca sexta (Lepidoptera, Sphingidae), a plasma membrane V-ATPase is present in the apical membranes of goblet cells where it energizes the alkalinization of the gut lumen to a pH of more than 11 (3). For the V 1 complex, amino acid sequences of five insect subunits A, B, E, F, and G have been deduced from cloned cDNAs (4), and evidence for the existence of subuni...

Genome-wide Analysis of Iron-dependent Growth Reveals a Novel Yeast Gene Required for Vacuolar Acidification

“…Sequence analysis of several Saccharomyces strains (22), as well as the experiments reported herein, demonstrate that Cwh36p is encoded by an intron-containing gene on the W-strand. The deduced protein shows homology to a small hydrophobic protein found in eukaryotes as diverse as insect (23) and cow (24). The protein was identified as a component of the H ϩ -ATPase, although its function remains unknown.…”

“…3A). Two of these proteins were identified because they were co-purified with the H ϩ -ATPase of insect vacuoles (23) and bovine Chromaffin granules (24). These proteins show two hydrophobic domains and have a hydropathy plot similar to Cwh36p (Fig.…”

The Yeast Vacuolar Proton-translocating ATPase Contains a Subunit Homologous to the Manduca sexta and Bovine e Subunits That Is Essential for Function

“…Significantly, the VMA21 gene showed some homology to the mammalian e subunit genes (8) but little or no homology to the M. sexta and yeast genes, and it was not grouped with the e subunits by the CDART program (26). The functional data presented here, along with the homology with the ϳ9-kDa V-ATPase-associated proteins from M. sexta and bovine cells (8,9) and the presence of homologs in many different eukaryotes, suggest that the e subunit may play an essential role in all V-ATPases.…”

“…3). Biochemical studies demonstrating association of homologous proteins with the M. sexta and bovine V-ATPases had suggested these proteins were subunits (8,9) but left open the possibility that they might be tissue-specific and provided no test of their significance in V-ATPase function. In contrast, the previously characterized yeast Vma21p is essential for V-ATPase function, based on the Vma Ϫ phenotype of vma21⌬ mutants, but is not part of the final assembled V-ATPase complex (10).…”

“…Although V-ATPase subunit composition is very similar among eukaryotes, highly purified preparations of the Manduca sexta and bovine chromaffin granule V-ATPases both contain membrane proteins of 9 -10 kDa that had not been observed in other systems, including yeast (8,9). This protein associated tightly with the V 0 sector in both preparations and appeared to be present at a comparable stoichiometry with the other V-ATPase subunits, prompting the investigators to name it the "e" subunit.…”