Download Isolation of the plc1 gene from the fission yeast

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Biochemical SocietyTransactions ( 1 995) 23
Isolation of the plcl gene from the fission yeast
Schizosaccharomyces pombe.
RITA SLAABY and JOHN DAVEY
School of Biochemistry, University of Birmingham,
Birmingham, B15 2lT,UK.
Conjugation between two haploid cells of the fission yeast
Schizosaccharomyces pombe involves a communication
system based on the reciprocal action of diffusible mating
pheromones (reviewed in [l]). Binding of the pheromones
to their specific receptors on the surface of the target cell
initiates a series of intracellular events leading to the
production of proteins responsible for the mating-specific
changes required for conjugation. The receptors are 7span proteins that couple to a heterotrimeric G protein
such that stimulation by pheromone causes the release of
the Galpha subunit. This acts as a positive regulator of the
signalling pathway and mediates activation of a series of
protein kinases that are functionally homologous to the
mitogen-activated protein (MAP) kinases involved in
controlling proliferation and differentiation in many higher
eukaryotes. The signalling pathway is reasonably well
understood but there is little information available
concerning events between the G protein and the kinase
cascade. Preliminary experiments have implicated the
activation of specific phospholipases in response to
pheromone addition [2] and we have used a PCR-based
approach to identify genes that encode these enzymes
(Figure 1). We now report the identification of p l c l , the
first S . p o m b e gene with the capacity to encode a
phopsholipase C (PLC). The putative Plcl protein has 899
amino acids and is structurally most similar to the 6 class of
PLC isozymes (Figure 2).
MNCEMVTSPESVNLGDSNRA
TLDNAALDLPYVGNRKKSEQ
SNSLSSFLSTKSTSASENKF
MNSARTNTSMNPYSCDSNEN
VPESIQNGCSLLRITKKKVR
201 KAYKKLCVDDIKEIRQCRDA
IYCADNKLKAMHMISPTLDA
IGLNLVCHQGEKMIDYSENL
RMCQMLHLNASMEFLEETFQ
LLKTRSEIVDIFKEYTSGSD
401 SDSIRTLYVSFCSNDDSKMG
1
VSPFCLPDCSENAVAQTRSK
DFFKMLSSRDRDAHSTLRKR
HGGLNWLSLKLNLLLRLQGR
LSTLSSVNQNFNSRQLLATI
QRKVSLDPISGYLMLDKNTG
RNYREQYKISSENEKRWFTI
HNQWIMALEGLKTYRLNEFI
NFWEKLEKEQSAQLDLGDVH
K
QHFVS EF-hand
K
L
X-region
601
Y -region
801
Figure 2 (above). Amino acid sequence of Plcl.
The putative Plcl protein contains 899 amino acids. The
conserved X and Y regions are highlighted and a possible
Ca2+-binding EF-hand is also indicated.
Figure 3 (below). Comparison of Plcl with other PLCs.
Comparisons of the X and Y regions from Plcl with
equivalent regions in each of the other PLCs are shown as
percentage identities. SH2 and SH3 indicate regions
homologous to the non-catalytic regions of the the src
gene product. The box just upstream of the X-region in
Plcl indicates the putative EF-hand motif.
X
S.pombe Plcl
Y
s.pOmbepkl- SSHNTYLLGKQFGGESSIEGYIRSLQRGCKCIEI
Primer 1
Primer 2
s.cerevisiae 6 - SSHNTYLLGKQIAETPSVEGYIQVLQQGCRCVEI
Bovine 62 . . . . . . . .V. D. .CGQS . . . . . .RA. KR. .... .V
Rat 61 ...........LTGPS.T.A..RA.CK....L ..
Bovinepl.......TAG.L.GNS . . .N.R...LS... ...L
Rat p1.......TAG.L.GNS...M.R ...LS ......L
H~manp2Dmsophila pl
Bovine yl
Rat yl -
.......TAG.FSGLS.A.M.R ...LS . . . . . .L
........S .R. .GGKS .. .M.R.T .LA. .....L
. . . . . . . .T.D. FSSES. L .A .ARC.RM. ...I.L
.......T.D.FSSES.L.A.ARC. RM.... 1.L
Figure 1. Cloning o f p f c l .
The catalytic domain of all PLC enzymes contains two
conserved regions (known as the X and Y regions) [3] and
the homology of the X-region (the amino acid sequence of
the X-region from several PLCs is shown as the single
letter code - residues identical to those in the S.cerevisiae
enzyme are indicated) was used to design two degenerate
oligonucleotide primers that would be expected to amplify
this region from S.pombe genomic DNA. A single PCRproduct was obtained and sequencing (the sequence of
the S.pombe product is shown at the top of the figure)
revealed significant homology to other members of this
family. This product was then used to isolate the complete
plcl gene from a library of S.pombe genomic DNA. For
original references and a more complete alignment, see [4].
S. cerevisiae
PLC 1
Rat PLC-61
200 a.a.
H
+ / H F }
Rat PLC-p1
Rat PLC-y1
47.2%
29.9%
SH2SH2 SH3
This work was supported by the Cancer Research
Campaign and the BBSRC. JD is a Lister Institute
Research Fellow.
The independent isolation ofplcl was recently reported [ 5 ] .
1. Nielsen, 0. & Davey, J. (1995) in Seminars in Cell Biology
(Davey, J.. ed.),vol6, pp 95-104, Academic Press,Camb.
2. Stuart, J., Hughes, P., Kirk, C.,Davey, J. & Michell. R.H.
(1995) Biochem. Soc. Trans. 23,223s
3. Rhee, S.G.& Choi, K.D. (1992) Adv. Second Messenger
Phosphoprot. Res. 26, 11-34
4. Flick, J.S. 8r Thomer, J. (1993) Mol. Cell. Biol. 13, 58615876
5 . Andoh, T., Yoko-0, T., Matsui, Y. & Toh-E, A. (1995)
Y-t
11, 179-185