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

Autor(en): Merzendorfer, H 
Huss, M 
Schmid, P
Harvey, WR
Wieczorek, H 
Stichwörter: Biochemistry & Molecular Biology; ENDOPLASMIC-RETICULUM; ESCHERICHIA-COLI; MANDUCA-SEXTA MIDGUT; PLASMA-MEMBRANE; POLYACRYLAMIDE GELS; POLYMERASE CHAIN-REACTION; PROTON PUMP; SECONDARY-STRUCTURE; TOBACCO HORNWORM MIDGUT; VACUOLAR-TYPE ATPASE
Erscheinungsdatum: 1999
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 274
Ausgabe: 24
Startseite: 17372
Seitenende: 17378
Zusammenfassung: 
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 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 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-0 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.
ISSN: 00219258
DOI: 10.1074/jbc.274.24.17372

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