Basic HTML Version
Molecular Soil Biology (online), 2011, Vol. 2 No.2, 9
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14
ISSN1925-2005
http://msb.sophiapublisher.com
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a kind of eukaryotic proteins’ posttranslational
modifications essential to their targeting (Apolloni et
al., 2000). These proteins end by the residues
recombination CAAX, named as CAAX proteins, and
their post-translational modifications usually include
the following three sequential, enzymatic steps. First,
the proteins are prenylated by one of two prenyl-
transferases named geranylgeranyltransferase
Ⅰ
or
farnesyltransferase (Galichet and Gruissem, 2003),
which happens in cytoplasm. In yeast and animal cells,
prenylation is followed by proteolytic removal of the
last three amino acids of the protein (AAX) by either
of the two endoproteases, RCE1 and STE24 (AFC1)
(Boyartchuk et al., 1997; Young et al., 2001), which is
thought to take place on the cytoplasmic surface of the
endoplasmic reticulum (ER) (Schmidt et al., 1998).
Finally, the exposed isoprenyl-cysteine is methylated
by and prenyl-dependent carbo-xylmethyltransferase
(PCM) (Clarke, 1992; Romano et al., 1998).
In the recent ten years, the protein prenylation in plant
has been clarified specifically, and genes encoding the
above enzymes have cloned in
Arabidopsis thilinana
.
There has been some reports showed that over-
expression of some genes is related to stress tolerance
of plant. In Arabidopsis, loss-of-function mutations in
the
ERA1
gene, encoding the β-subunit of PFT,
ggb1
gene, encoding the β
-
subunit of PGGT
Ⅰ
, or
plp
gene,
which encode α
-
subunit of these two enzymes, cause
an enhanced response to abscisic acid (ABA) in seed
germination and stomatal closure assays (Cutler et al.,
1996; Pei et al., 1998; Running et al., 2004; Johnson
et al., 2005). The above two enzymes involved in
negative regulation of signaling in guard cells.
AtSTE24,
an Arabidopsis homologue of the CAAX
protease STE24, was cloned and expressed in
rce1
∆
ste24
∆ mutant yeast to demonstrate functional
complementation (Bracha et al., 2002). To date, there
are few studies were reported on AtSTE24, and fewer
reports introducing its relationship with stresses
tolerance and responsion reaction with metal ions.
This paper reports on the cloning and characterization
of
PutSTE24
and
AtSTE24
, indicating that STE24 is a
protease related to Al
3+
tolerance and other stresses in
yeast.
1 Results and Analysis
1.1 Cloning and sequence analisys of
PutSTE24
In the previous studies, full length cDNAs over-
expressing library of
Puccinellia tenuifolra
was
constructed in yeast (
Saccharomyces cerevisiae
). A
clone was screened out from this yeast library with
medium containing AlCl
3
. By PCR using the specific
primers described in materials and methods and
sequencing, results showed that
PutSTE24
cDNA
contained full length of 1 700 nucleotides and had a
open reading frame (ORF) of 1 275 bp nucleotides
encoding a predicted 424 amino acids (Figure 1). The
predicted protein was calculated to have a molecular
mass of 48.3 kD and pI of 6.84.
The Blast algorithm identified three proteins with
higher similarity to PutSTE24 (Figure 2). They are
AtSTE24 from
Arabidopsis thaliana
(At4g01320,
77% amino acid identity), CAAX prenyl protease 1
from
Zea mays
(100286144, 79% amino acid identity),
and putative STE24 from
Ricinus communis
(8286673,
77% amino acid identity). Like AtSTE24, PutSTE24
possesses two conservative sequence motifs: HEXXH
that is a signature of zinc metalloproteases and a
C-terminal KKXX, the ER membrane retention signal
(Figure 2).
1.2 Over-expressing of
PutSTE24
and
AtSTE24
respectively in yeast and Al
3+
tolerance analysis
In this study,
PutSTE24
was screened out with AlCl
3
stress, therefore, to further analyze the responsive
relationship of it and its homologue
AtSTE24
with
Al
3+
stress, yeast transformed lines were constructed.
One was transformed with empty vector pAUR123 as
a control. The two else transformants were
over-expressed
PutSTE24
and
AtSTE24
respectively
(Figure 3; Figure 4 and Figure 5). In the presence of
different concentrations of AlCl
3
, the growth of these
transformants showed differently (Figure 3). The
growth of these two transformants showed similarly.
At 6 mmol/L of AlCl
3
, they grew much better than the
control yeast; but at 6.5 or 7 mmol/L of AlCl
3
stress,
this growth advantage disappeared, and they seemed
similar to the control, even worse. The results
indicated over-expressing of
PutSTE24
and
AtSTE24
can alleviate Al
3+
stress at a degree.