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Molecular cloning of cDNA encoding the 16 KDa subunit of vacuolar H(+)-ATPase from mouse cerebellum
H Hanada1, M Hasebe, Y Moriyama
1Department of Organic Chemistry and Biochemistry, Osaka University, Japan.
Abstract:
cDNA for the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from mouse cerebellum and sequenced. The deduced polypeptide (155 amino acid residues; molecular weight, 15,808) was highly hydrophobic and homologous to the subunits of bovine adrenal medulla, Torpedo marmorata electric lobe, Drosophila and yeast. Glu-139 (supposed to be essential for proton transport) was also conserved as the potential dicyclohexylcarbodiimide binding site. The subunit had four transmembrane segments: Segment II and IV were highly homologous and Glu-139 was located in Segment IV. The roles of the non-conserved regions are discussed.
Insights
Researchers cloned and sequenced the 16 kDa subunit of mouse vacuolar H(+)-ATPase, revealing its hydrophobic nature and conserved functional sites essential for proton transport.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Vacuolar H(+)-ATPase (V-ATPase) is a crucial proton pump found in various organisms.
- Understanding the structure and function of V-ATPase subunits is vital for cellular physiology.
Purpose of the Study:
- To clone and sequence the cDNA encoding the 16 kDa subunit of mouse vacuolar H(+)-ATPase.
- To analyze the deduced polypeptide's structural and functional characteristics.
- To compare the mouse subunit with homologous subunits from other species.
Main Methods:
- cDNA cloning from mouse cerebellum.
- DNA sequencing.
- Bioinformatic analysis of the deduced polypeptide sequence.
Main Results:
- The 16 kDa subunit cDNA was successfully cloned and sequenced from mouse cerebellum.
- The deduced polypeptide is 155 amino acids with a molecular weight of 15,808 Da and is highly hydrophobic.
- Homology was observed with V-ATPase subunits from bovine, Torpedo, Drosophila, and yeast, including conserved Glu-139, a potential proton transport and DCCD binding site.
- Four transmembrane segments were identified, with segments II and IV showing high homology and Glu-139 located in segment IV.
Conclusions:
- The mouse 16 kDa V-ATPase subunit shares significant structural and functional conservation with orthologs from diverse species.
- The conserved Glu-139 residue is likely critical for proton transport and dicyclohexylcarbodiimide binding.
- The identified transmembrane segments suggest a role in membrane integration and proton translocation.
- Further investigation into non-conserved regions may elucidate specialized functions.