Evidence that the 16 kDa proteolipid (subunit c) of the vacuolar H(+)-ATPase and ductin from gap junctions are the same polypeptide in Drosophila and Manduca: molecular cloning of the Vha16k gene from Drosophila.
The 16 kDa proteolipid (subunit c) of the eukaryotic vacuolar H(+)-ATPase (V-ATPase) is closely related to the ductin polypeptide that forms the connexon channel of gap junctions in the crustacean Nephrops norvegicus. Here we show that the major protein component of Manduca sexta gap junction preparations is a 16 kDa polypeptide whose N-terminal sequence is homologous to ductin and is identical to the deduced sequence of a previously cloned cDNA from Manduca (Dow et al., Gene, 122, 355-360, 1992). We also show that a Drosophila melanogaster cDNA, highly homologous to the Manduca cDNA, can rescue Saccharomyces cerevisiae, defective in V-ATPase function, in which the corresponding yeast gene, VMA3, has been inactivated. Evidence is presented for a single genetic locus (Vha16) in Drosophila, which in adults at least contains a single transcriptional unit. Taken together, the data suggest that in Drosophila and Manduca, the same polypeptide is both the proteolipid subunit c component of the V-ATPase and the ductin component of gap junctions. The intron/exon structure of the Drosophila Vha16 is identical to that of a human Vha16 gene, and is consistent with an ancient duplication of an 8 kDa domain. A pilot study for gene inactivation shows that transposable P-elements can be easily inserted into the Drosophila ductin Vha16 gene. Although without phenotypic consequences, these can serve as a starting point for generation of null alleles.
Finbow
M E
ME
CRC Beatson Laboratories, Beatson Institute for Cancer Research, Bearsden, Glasgow, Scotland.
Goodwin
S F
SF
Meagher
L
L
Lane
N J
NJ
Keen
J
J
Findlay
J B
JB
Kaiser
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017630:Connexins / Q000235:genetics; D003445:Crustacea / Q000378:metabolism; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D005091:Exons; D017629:Gap Junctions / Q000201:enzymology* / Q000648:ultrastructure; D017344:Genes, Insect; D006801:Humans; D007438:Introns; D046911:Macromolecular Substances; D018402:Manduca / Q000201:enzymology* / Q000235:genetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011510:Proteolipids / Q000096:biosynthesis / Q000235:genetics*; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases
D017630:Connexins; D017931:DNA Primers; D046911:Macromolecular Substances; D011510:Proteolipids; D012333:RNA, Messenger; C082471:ductin; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases