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.

REFERENCES

Apperson
M.
,
Jensen
R. E.
,
Suda
K.
,
Witte
C.
,
Yaffe
M. P.
(
1990
).
A yeast protein, homologous to the proteolipid of the chromaffin granule proton ATPase, is important for cell growth.
Biochem. Biophys. Res. Commun
168
,
574
579
.
Ballinger
D. G.
,
Benzer
S.
(
1989
).
Targeted gene mutations in Drosophila.
Proc. Nat. Acad. Sci. USA
86
,
9402
9406
.
Bohrmann
J.
(
1993
).
Antisera against a channel-forming 16 kDa protein inhibit dye-coupling and bind to cell membranes in Drosophila ovarian follicles.
J. Cell Sci
105
,
513
518
.
Buultjens
T. E. J.
,
Finbow
M. E.
,
Lane
N. J.
,
Pitts
J. D.
(
1988
).
Tissue and species conservation of the vertebrate and arthropod forms of the low molecular weight (16–18000) proteins of gap junctions.
Cell Tiss. Res
251
,
571
580
.
Chomczynski
P.
,
Saachi
N.
(
1987
).
Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem
162
,
156
159
.
Dow
J. A. T.
,
Goodwin
S. F.
,
Kaiser
K.
(
1992
).
Analysis of the gene encoding a 16-kDa proteolipid subunit of the vacuolar H+-ATPase from Manducasexta midgut and tubules.
Gene
122
,
355
360
.
Finbow
M. E.
,
Buultjens
T. E. J.
,
Lane
N. J.
,
Shuttleworth
J.
,
Pitts
J. D.
(
1984
).
Isolation and characterisation of arthropod gap junctions.
EMBO J
3
,
2271
2278
.
Finbow
M. E.
,
Eliopoulos
E. E.
,
Jackson
P. J.
,
Keen
J. N.
,
Meagher
L.
,
Thompson
P.
,
Jones
P.
,
Findlay
J. B. C.
(
1992
).
Structure of a 16kD protein that has identity to the putative proton channel of the vacuolar H+-ATPase.
Protein Eng
5
,
7
15
.
Finbow
M. E.
,
John
S.
,
Kam
E.
,
Apps
D. K.
,
Pitts
J. D.
(
1993
).
Disposition and orientation of ductin (DCCD reactive vacuolar H+-ATPase subunit) in mammalian membrane complexes.
Exp. Cell Res
207
,
261
270
.
Finbow
M. E.
,
Pitts
J. D.
(
1993
).
Is the gap junction channel-the connexon-made of connexin or ductin?.
J. Cell Sci
106
,
463
472
.
Gillespie
G. A.
,
Somlo
S.
,
Germino
G. G.
,
Weinstat-Swallow
D.
,
Reeders
S. T.
(
1991
).
CpG islands in the region of an autosomal dominant polycystic kidney disease locus defines the 5end of a gene encoding a putative proton channel.
Proc Nat. Acad. Sci. USA
88
,
4289
4293
.
Guthrie
C.
,
Fink
G. R.
(
1991
).
Guide to yeast genetics and molecular biology.
Meth. Enzymol.
194
Harrison
M. A.
,
Jones
P. C.
,
Kim
Y.-I.
,
Finbow
M. E.
,
Findlay
J. B. C.
(
1994
).
Functional properties of a hybrid vacuolar H+-ATPase in yeast cells expressing the Nephrops 16-kDa proteolipid.
Eur. J. Biochem
221
,
111
126
.
Holzenburg
A.
,
Jones
P. C.
,
Franklin
T.
,
Pali
T.
,
Heimburg
T.
,
Marsh
D.
,
Findlay
J. B. C.
,
Finbow
M. E.
(
1993
).
Evidence for a common structure for a class of membrane channel.
Eur. J. Biochem
213
,
21
30
.
Kaiser
K.
(
1990
).
From gene to phenotype in Drosophila and other organisms.
BioEssays
12
,
297
301
.
Kaiser
K.
,
Goodwin
S. F.
(
1990
).
‘Site selected’ transposon mutagenesis in Drosophila.
Proc. Nat. Acad. Sci. USA
87
,
1686
1690
.
Lane
N. J.
,
Dilworth
S. M.
(
1989
).
Isolation and biochemical characterization of septate juntions: differnces between the proteins in smooth and pleated varieties.
J. Cell Sci
93
,
123
131
.
Mandel
M.
,
Moriyama
Y.
,
Hulmes
J. D.
,
Pan
Y.-C.
,
Nelson
H.
,
Nelson
N.
(
1988
).
cDNA sequence encoding the 16-kDa proteolipid ofchromaffin granules implies gene duplication in the evolution of H+-ATPases.
Proc. Nat. Acad. Sci. USA
85
,
5521
5524
.
Marsh
J. L.
,
Erfle
M.
,
Wykes
E. J.
(
1984
).
The pIC plasmid and phage vectors with versatile cloning site for recombinant selection by insertional inactivation.
Gene
32
,
481
485
.
Meagher
L.
,
McLean
P.
,
Finbow
M. E.
(
1990
).
Sequence of a cDNA from Drosophila coding for the 16-kDa component of the vacuolar H+-ATPase.
Nucl. Acids Res
18
, 6712-.
Nelson
H.
,
Nelson
N.
(
1990
).
The progenitor of ATP synthases was closely related to the current vacuolar H+-ATPase.
FEBS Lett
247
,
147
153
.
Nelson
H.
,
Nelson
N.
(
1990
).
Disruption of genes encoding subunits of yeast vacuolar H+-ATPase causes conditional lethality.
Proc. Nat. Acad. Sci. USA
87
,
3503
3507
.
Nelson
N.
(
1992
).
Organellar proton-ATPases.
Curr. Opin. Cell Biol
4
,
654
660
.
Nezu
J.-I.
,
Motojima
K.
,
Tamura
H.-i.
,
Ohkuma
S.
(
1992
).
Molecular cloning of a rat liver cDNA encoding the 16 kDa subunit of the vacuolar H+-ATPase: organellar and tissue distribution of 16 kDa proteolipids.
J. Biochem
112
,
212
219
.
Noumi
T.
,
Beltran
C.
,
Nelson
N.
(
1991
).
Mutational analysis of yeast vacuolar H+-ATPase.
Proc. Nat. Acad. Sci. USA
88
,
1938
1942
.
O'Hare
K.
,
Rubin
G. M.
(
1983
).
Structure of P transposable elements and their sites of excision in the Drosophila melanogaster genome.
Cell
34
,
25
35
.
Robertson
H. M.
,
Preston
C. R.
,
Phillis
R. W.
,
Johnson-Schlitz
D. M.
,
Benz
W. K.
,
Engels
W. R.
(
1988
).
A stable source of P-element transposase in Drosophila melanogaster.
Genetics
118
,
461
470
.
Ryerse
J. S.
(
1989
).
Isolation and characterization of gap junctions isolated from Drosophila melanogaster.
Cell Tiss. Res
256
,
7
16
.
Ryerse
J. S.
(
1989
).
Electron microscope immunolocalization in Drosophila.
Tissue & Cell
21
,
835
839
.
Ryerse
J. S.
(
1991
).
Gap junction-associated protein: tissue distribution and abundance in the adult brain of Drosophila.
Tissue& Cell
23
,
709
718
.
Ryerse
J. S.
(
1993
).
Structural, immunocytochemical, and initial biochemical characterization of NaOH extracted gap junctions from an insect, Heliothis viriscens.
Cell Tiss. Res
274
,
393
404
.
Segalat
L.
,
Perichon
R.
,
Bouly
J.-P.
,
Lepesant
J. A.
(
1992
).
The Drosophilapourquoi-pas/wingsdown zinc finger protein: oocyte nucleus localization and embryonic requirement.
Genes Dev
6
,
1019
1029
.
Umemoto
N.
,
Yoshihisa
T.
,
Hirata
R.
,
Anraku
Y.
(
1990
).
Roles of the VMA3 gene product, subunit c of the vacuolar membrane H+-ATPase on vacuolar acidification and protein transport.
J. Biol. Chem
265
,
18447
18453
.
Umemoto
N.
,
Ohya
Y.
,
Anraku
Y.
(
1991
).
VMA11, a novel gene that encodes a putative proteolipid, is indispensable for expression of yeast vacuolar membrane H+-ATPase activity.
J. Biol. Chem
266
,
24526
24532
.
Winship
P. R.
(
1989
).
An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide.
Nucl. Acids Res
17
, 1266-.
This content is only available via PDF.