Worm Breeder's Gazette 15(3): e2 (June 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Laboratory of Biochemistry III, Swiss Federal Institute of Technology (ETH),, Zurich, Switzerland
Calcium-pumping ATPases and sodium-calcium exchangers play an important
role in intracellular calcium homeostasis of excitable cells in mammals
(1,2). In mammalian genomes these proteins are encoded by gene
families, individual members of which feature complex tissue-specific
expression and alternative splicing. Genes of these transporters are
large (>100 kb) and are regulated by multipartite promoters (3, 4). In
search for a less complex system which is more amenable to genetic
manipulation, we intend to identify a complete set of nematode genes,
encoding calcium ATPases, Na/Ca- and Na/Ca,K-exchangers, using Genome
Project data as a starting point. At this time we report cloning and
functional expression of the ORF W09C2.3, which encodes a
calcium-pumping ATPase (cemca1) with the features resembling the
relatively well studied PMCA family of mammalian cells.
The protein was originally identified as a Wormpep entry showing
about 65% similarity to PMCA family ATPases. ORF W09C2.3, encoding the
protein, corresponds to a group of relatively abundant ESTs , of which
the clone yk49f8 (5) was found to contain about 70% of the coding
sequence. The missing part of the coding sequence was obtained by RT-PCR
on polyA-RNA isolated from strain Bristol of C.elegans (6), using a
SL1-specific primer and a gene-specific primer. In the course of this
experiment it was found that the N-terminus of the protein consists of
four variants, generated by alternative splicing of 3 alternative first
exons, each of which appends a short amino acid sequence to the fourth
("basic") form, encoded in exon 2 and the exons downstream of it. The
cDNA encoding the shortest form of 1202 aminoacids (131 kDa) was
modified by PCR to include a FLAG tagging sequence at the C-terminus,
was subsequently inserted into a baculovirus vector and introduced into
Sf9 insect cells by lipotransfection.
The crude membrane preparation from cells, infected with the
recombinant virus show a specific band of 130 kDa, reacting with
anti-FLAG M2 monoclonal antibody. The same membrane preparation, when
introduced into the reaction of the phosphorylated intermediate
formation shows a pattern (stimulation of the 130 kDa band by calcium
plus lanthanum, inhibition by EGTA) indicative of a plasma membrane
calcium ATPase.
Using a similar approach, we have identified a family of 3
genes, encoding putative Na/Ca-exchangers, similar to the mammalian NCX
family, and a family of 2 genes, encoding putative potassium-dependent
Na/Ca-exchangers. All transporter transcripts that are completed begin
with the SL1 sequence, indicating that they are solitary transcripts or
first genes in an operon. The proteins they encode can be modelled with
a typical "mammalian" topology (5 transmembrane domains-cytoplasmic
loop-6 transmembrane domains). Like their mammalian counterparts, some
of them undergo alternative splicing in a distal portion of the
cytoplasmic loop. Experiments are in progress to demonstrate the
functional activity of these proteins.
References:
1. Philipson, K.D. and Nicoll, D.A. (1993) Int. Rev.Cytol. 137c,
pp.199-227.
2. Carafoli, E. (1992) J.Biol.Chem., 267, pp.2115-2118.
3. Hilfiker, H., Strehler-Page, M.-A., Stauffer, T.P. Carafoli, E. and
Strehler, E.E. (1993) J.Biol.Chem., 268, pp. 19717-19725.
4. Scheller, T., Kraev, A., Skinner, S. and Carafoli, E. (1998)
J.Biol.Chem. 273, pp. 7643-7649.
5. This and other cDNA clones obtained from Y.Kohara, National Institute
of Genetics, Japan.
6. Obtained from T.Stiernagle, Caenorhabditis Genetics Centre, USA.