Abstract
To understand the molecular structure of the vacuolar H(+)-translocating ATPase from plants, cDNAs encoding the N,N'-dicyclohexylcarbodiimide-binding 16-kDa proteolipid from oat (Avena sativa L. var. Lang) have been obtained. A synthetic oligonucleotide corresponding to a region of the bovine proteolipid cDNA (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-C.E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5521-5524) was used to screen an oat cDNA library constructed in lambda gt11. The nucleotide sequences of several positive clones (VATP-P1, clones 12, 54, 93) demonstrated the presence of a small multigene family. The four clones showed extensive divergence in their codon usage and their 3'-untranslated regions; however, the deduced amino acid sequences of the proteins were 97-99% identical. These clones encoded the proteolipid subunit as one of them (clone 12) expressed a fusion protein that reacted with an antibody to the 16-kDa proteolipid. The open reading frame of one cDNA clone (VATP-P1) predicted a polypeptide of 165 amino acids with a molecular mass of 16,641. Based on hydropathy plots, a molecule with four membrane-spanning domains was predicted, in which domain IV was especially conserved among different species. This domain showed 80% identity in nucleotide or amino acid sequences between the oat and the bovine proteolipids and contained a glutamate residue that is the putative N,N'-dicyclohexylcarbodiimide-binding residue. The presence of a small multigene family of the 16-kDa proteolipid was confirmed by Southern blot analysis showing that several distinct restriction fragments of oat nuclear DNA hybridized with the VATP-P1 cDNA.
Highlights
Molecular Cloning and Sequencing of cDNAs Encoding the Proteolipid Subunit of the Vacuolar H+-ATPase froma Higher Plant*
Isolation of cDNA Clones-Our initial strategy for cloning the cDNA encoding the proteolipid of oat V-ATPase was to construct an oat cDNA library in Xgtll
To ensure the presence of V-ATPase cDNAs we constructed the cDNA library using a poly(A)+RNA preparation of oat roots from which VATPase subunits could be translated in uitro and identified by immunoprecipitation [29]
Summary
Plant Material-Oat seeds (Avena sativa L. var. Lang) were germinatedin the dark (22 "C) over an aerated solution of0.5 mM CaS04.Roots were harvested after 3 days of growth. The poly(A)+RNA preparation from oat roots was translated in a rabbit reticulocyte lysate (Promega) with labeling of [35S]methionine(Du Pont-New England Nuclear). Translation products at molecular masses of kDa, and occasionally at 16 kDa were detected as shown before [29] Such a poly(A)+RNA preparation was used for construction of a cDNA library. CDNA inserts were cleaved from recombinant phage DNA with EcoRI, separated by agarose gel electrophoresis, and recovered by electrophoresis onto DEAE-nitrocellulose membrane (NA-45, Schleicher & Schuell). 18 DNA (Bethesda Research Laboratories) and synthetic primers (20-30 bases) to internalsequences of the cDNA inserts (see Fig. 2A legend) were used to obtain the sequences of both strands. Nuclear DNA Isolation and Southern Blotting-The nuclear DNA from leaves of 7-day-old-oat seedlings was isolated as described [33], digested with EcoRI, BamHI, or HindIII, separated by 1.0% agarose gel electrophoresis, and transferreodnNtoytran membrane (Schleicher & Schuell). 0.1 X SSPE and0.5% SDS for 45 min
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
