A novel insect V-ATPase subunit M9.7 is glycosylated extensively

Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypep tide. Based on N-terminal protein sequencing, we cloned a corresponding cDN A. The deduced hydrophobic protein consisted of 88 amino acids with a molec ular mass of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypepti de, using a monoclonal antibody to the 20-kDa polypeptide, confirmed that t he correct cDNA had been cloned. The 20-kDa polypeptide is glycosylated, as deduced from lectin staining. Treatment with N-glycosidase A resulted in t he appearance of two additional protein bands of 16 and 10 kDa which both w ere immunoreactive to the 20-kDa polypeptide-specific monoclonal antibody. Thus, extensive N-glycosylation of the novel V-0 subunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophores is. M9.7 exhibits some similarities to the yeast protein Vma21p which resid es in the endoplasmic reticulum and is required for the assembly of the V-0 complex. However, as deduced from immunoblots as well as from activities o f the V-ATPase and endoplasmic reticulum marker enzymes in different membra ne preparations, M9.7 is, in contrast to the yeast polypeptide, a constitut ive subunit of the mature plasma membrane V-ATPase of M. sexta.