From the Gladstone Institute of Cardiovascular
Disease, the § Cardiovascular Research Institute, and the
Department of Medicine, University of California, San Francisco,
California 94141-9100; the ** Department of Pharmacology and Cancer
Biology, Duke University Medical Center, Durham, North Carolina
27710-3686; and the
Molecular Genetics
Section, Division of Biomedical Sciences, Imperial College School of
Medicine, Sir Alexander Fleming Building, London, SW7 2AZ, United
Kingdom
Received for publication, November 26, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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After isoprenylation, Ras and other
CAAX proteins undergo endoproteolytic processing by Rce1
and methylation of the isoprenylcysteine by Icmt (isoprenylcysteine
carboxyl methyltransferase). We reported previously that
Rce1-deficient mice died during late gestation or soon
after birth. We hypothesized that Icmt deficiency
might cause a milder phenotype, in part because of reports suggesting the existence of more than one activity for methylating isoprenylated proteins. To address this hypothesis and also to address the issue of
other methyltransferase activities, we generated
Icmt-deficient mice. Contrary to our expectation,
Icmt deficiency caused a more severe phenotype than
Rce1 deficiency, with virtually all of the knockout embryos
(Icmt After isoprenylation, Ras and other proteins that terminate
with a CAAX1
sequence undergo two additional C-terminal modifications (1). First,
the last three amino acids of the protein (i.e. the
-AAX) are released by an endoprotease associated with the
endoplasmic reticulum (1-3). Second, the carboxyl group of the newly
exposed isoprenylcysteine is methylated (4, 5). These
post-isoprenylation processing steps may help target CAAX
proteins to membrane surfaces within cells (1).
The endoprotease and methyltransferase steps have attracted interest
because they offer a potential means for modulating the activity of
CAAX proteins, many of which participate in cell signaling (1). Several groups have hypothesized that inhibiting the endoprotease or the methyltransferase might retard the growth of tumors caused by
mutation-activated Ras proteins (1, 2, 6, 7). At this point, however,
testing such hypotheses appears to be a few years away. No specific
high affinity inhibitors suitable for animal testing have been
developed, either for the endoprotease or for the methyltransferase.
Just as importantly, neither the spectrum of substrates nor the
physiologic importance of the two processing steps has been explored
fully. This is particularly the case for the methyltransferase.
To define the physiologic relevance of the post-isoprenylation
processing steps, our laboratory generated and characterized mice
lacking the endoprotease Rce1 (8). Membranes from
Rce1-deficient embryos and cells were completely unable to
carry out the endoproteolytic processing of Ras and a host of other
CAAX proteins. Surprisingly, the consequences of knocking
out Rce1 in the mouse were relatively mild. Although most of
the Rce1 knockout mice died before birth, the embryos
remained viable until late in gestation, and as late as embryonic day
18.5 many were normal in size, appeared healthy, and had no obvious
histologic abnormalities. A few of the Rce1 knockout mice
were born and lived for a few weeks.
A methyltransferase for mammalian CAAX proteins,
isoprenylcysteine carboxyl methyltransferase (Icmt), has been
identified recently (5) and shown to be located in the endoplasmic
reticulum (5, 9). We used gene-targeting techniques to produce a mouse embryonic stem (ES) cell line lacking both Icmt alleles and
documented that membranes from those cells lacked the ability to
methylate recombinant K-Ras (10). It is important to note, however,
that existing reports have raised the possibility that certain
isoprenylated proteins might be methylated by other enzymatic
activities, at least in some cell types. For example, Giner and Rando
(11) concluded that there were distinct methyltransferase activities for the two classes of carboxyl-methylated isoprenylated proteins, the
CAAX proteins and the CXC Rab proteins (11). That
conclusion was based on a variety of reciprocal inhibition studies with
different methyltransferase substrates and inhibitors.
The goal of the current study was to generate Icmt knockout
mice, both to define the physiologic consequences of Icmt
deficiency and to further define biochemical roles of Icmt. Based in
part on the report of an additional methyltransferase activity (11), our a priori prediction was that Icmt-deficient
mice might be affected less severely than the Rce1-deficient
mice and might even be viable. We also predicted that
Icmt-deficient cells might retain the capacity to methylate
the CXC-containing Rab proteins. As outlined in this report,
both of those expectations were dashed by our experimental results.
Generation of Icmt-deficient Mice--
A sequence replacement
gene-targeting vector designed to replace exon 1 of the mouse
Icmt gene with a neomycin-resistance gene (10) was
electroporated into 129/SvJae ES cells, and targeted cells
(Icmt+/ Production of Chimeric Mice from Icmt Measurement of Icmt Activity in Embryo Lysates--
Embryos were
harvested and immediately placed in ice-cold buffer A (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 100 mM NaCl), supplemented with a
protease inhibitor mixture (Complete Mini, Roche Molecular
Biochemicals). The embryos were homogenized with a Polytron and then
centrifuged at 500 × g for 5 min to remove debris. The
protein concentration of the homogenate was determined with a Bio-Rad
DC Protein Assay (Bio-Rad, Hercules, CA). To measure Icmt activity,
lysates (40-100 µg) were incubated with 10 µM
S-adenosyl-L-[methyl-14C]methionine
(55 Ci/mol, Amersham Pharmacia Biotech) and 50 µM of
either
N-acetyl-S-geranylgeranyl-L-cysteine
(AGGC, Biomol) or
N-acetyl-S-farnesyl-L-cysteine (AFC,
Biomol). Recombinant farnesyl-K-Ras (7) and recombinant
geranylgeranyl-Rab proteins (described below) were also tested as
methyl-accepting substrates. The total volume for the methylation
reactions was 50 µl. After a 30-min incubation at 37 °C, the
methylation reaction was stopped by adding 50 µl of 1.0 M
NaOH containing 0.1% SDS. Most of the reaction mixture (90 µl) was
spotted onto a pleated 2 × 8-cm filter paper wedged in the neck
of a 20-ml scintillation vial containing 5 ml of scintillation fluid
(ScintiSafe Econo 1, Fisher). The vials were capped and incubated at
room temperature for 5 h to allow the [14C]methanol
(formed by base hydrolysis of methyl esters) to diffuse into the
scintillation fluid (4). The filter papers were then removed, and the
vials were counted for radioactivity. Methyltransferase activity
(pmol/mg total cell protein/min) was calculated after subtracting the
background level of methylation in control reactions (lysates and
S-adenosyl-L-[methyl-14C]methionine
but no isoprenylated substrates). For the Rab methylation assays,
control reactions also contained Rab1A, a CC Rab protein that does not
undergo carboxyl methylation (12).
Expression and Purification of Recombinant Rab
Proteins--
Recombinant Rab proteins, Rab escort protein (REP), and
Rab geranylgeranyltransferase (RabGGTase) were purified as previously described (13-15). Briefly, bovine Rab3B, murine Rab3D, and human Rab6
were expressed as N-terminal histidine-tagged fusion proteins in
Escherichia coli with pET14b (Novagen, Madison, WI) as the expression vector for Rab3B and Rab6 and pRSET (Invitrogen, Carlsbad, CA) for Rab3D. The proteins were purified by affinity chromatography using a Ni2+-Sepharose resin. RabGGTase was expressed in
Sf9 insect cells by coinfection with baculoviruses coding for
both the Preparation of Gernanylgeranylated REP·Rab
Complexes--
Geranylgeranylated Rab·REP complexes (REP·RabGG)
were formed in vitro by incubating Rab proteins with
RabGGTase, and geranylgeranylpyrophosphate (GGPP) in the presence of
limiting amounts of REP (16). Rab protein (10 µM) was
incubated with 2.5 µM REP1, 0.7 µM
RabGGTase, and 20 µM unlabeled GGPP (Sigma) in a 50 mM sodium HEPES buffer, pH 7.2, containing 5 mM
MgCl2, 1 mM dithiothreitol, and 0.05 mM protein-grade Nonidet P-40 (Calbiochem). The 50 µl
reaction was incubated at 37 °C for 45 min. The complexes were
stored at 4 °C with 1 mg/ml of bovine serum albumin as a carrier
protein. To determine the efficiency of geranylgeranylation, parallel
reactions were performed in the presence of [3H]GGPP
([1-3H] all-trans-GGPP, 15-30 Ci/mmol,
PerkinElmer Life Sciences), and the incorporated [3H]GG
was measured by scintillation after filtration through glass fiber
filters (15).
Northern Blot Analysis--
A 262-base pair
32P-labeled Icmt cDNA probe (spanning exons
1-3 of the Icmt gene) was hybridized to a mouse
multiple-tissue poly(A)+ RNA blot (CLONTECH,
Palo Alto, CA); hybridization and washing were performed as described
previously (7). The blot was exposed to x-ray film for 72 h at
Quantification of Substrate Accumulation in Icmt Biochemical Analysis of Icmt-deficient
Embryos--
Icmt+/
To gain insights into the importance of Icmt in the
formation of different organs, we generated male chimeric mice
(n = 10) with two lines of Icmt
To determine whether Icmt
Consistent with the apparent absence of a redundant methyltransferase
activity, there was a substantial accumulation of methyltransferase substrates in lysates from Icmt
An earlier study (11) concluded that distinct
S-adenosylmethionine-dependent methyltransferase
activities were responsible for the methylation of the CAAX
and CXC groups of isoprenylated proteins. That result would
predict that lysates from Icmt Icmt catalyzes the formation of a carboxyl methyl ester on the
isoprenylcysteine of CAAX proteins. The methylation reaction is the last of three sequential CAAX-box modifications and
the most subtle, at least from the perspective of the primary structure of the protein. Methylation changes the molecular mass of the protein
by a mere 14 daltons versus several hundred for both the isoprenylation and endoprotease steps. We had predicted that
Icmt-deficiency might produce a relatively mild phenotype.
First, deletion of the methyltransferase gene in yeast (i.e.
STE14) has little impact apart from its effect on the mating
pheromone a-factor (17), and a-factor apparently does not exist in
mammals. Second, Rce1 deficiency produced a relatively mild
phenotype, with some knockout mice surviving for a few weeks after
birth. Thus, we expected that murine Icmt deficiency would
produce a similarly mild phenotype, or perhaps even milder given the
studies suggesting the existence of additional Icmt-like
activities in mammalian cells (11, 18). Our a priori
prediction was not upheld. Icmt deficiency yielded a more
severe phenotype, with most Icmt The Icmt Why were the developmental abnormalities more severe in
Icmt Our current studies show that the methylation of the CXC Rab
proteins is carried out by Icmt, the same methyltransferase that is
responsible for CAAX protein methylation. Thus, Icmt almost certainly has more substrates than Rce1, which has no role in Rab
protein processing. If these additional substrates (i.e. the Rab proteins) lie at the root of the more severe developmental defects
in Icmt deficiency, one might predict that the methylation of Rab
proteins has a significant impact on their function. In the case of
Rab6, we did not observe a detectable effect of methylation on the
intracellular localization of the protein, but we caution against
overinterpreting those results. Those experiments did not assess other
potential effects of Rab methylation such as effects on protein
function or stability.
A second potential explanation for the more severe developmental
problems in the Icmt The positioning of the carboxylate anion might also affect
prenylation-dependent protein-protein interactions. Chen
et al. (26) have shown that the binding of K-Ras to
microtubules is dependent on the structure of the C terminus.
Farnesylated K-Ras binds to microtubules, but this binding is
eliminated by the Rce1-mediated release of the last three amino acids.
Binding to microtubules is restored by carboxyl methylation. Thus, in
the case of K-Ras, the precise structure of the C terminus affects
protein-protein interactions. These data, along with the aforementioned
considerations regarding membrane binding, have made the "carboxylate
anion positioning" hypothesis quite intriguing. Of course, the effect
of carboxylate anion positioning could differ for different
CAAX proteins.
A third potential explanation for the more severe phenotype in
Icmt-deficient mice compared with Rce1-deficient
mice is that some CAAX proteins might undergo
endoproteolytic processing in the absence of Rce1. According
to this hypothesis, the milder phenotype of Rce1 deficiency
could reflect the fact that Rce1 processes fewer CAAX
protein substrates than Icmt. This possibility is reasonable, given
that a second yeast protein, Afc1p (Ste24p),3 assists Rce1p
in the removal of the -AAX from the farnesylated mating
pheromone a-factor. Thus far, however, there has been no direct
demonstration that the murine ortholog of AFC1,
Zmpste24,3 has any role in CAAX
protein processing (1).
The development and characterization of Icmt-deficient mice
has clarified the role of Icmt in mouse development and in
the processing of isoprenylated proteins. Just as importantly, these studies have suggested new hypotheses. For example, it will now be of
interest to determine the importance of methylation in Rab protein
function and to test the carboxylate anion positioning hypothesis. In addition, the production of Icmt-deficient
fibroblasts opens the door to addressing the importance of carboxyl
methylation in the transformation of fibroblasts by mutation-activated
forms of the Ras proteins.
/
) dying by mid-gestation. An analysis of chimeric
mice produced from Icmt
/
embryonic stem cells showed that the Icmt
/
cells retained the capacity to
contribute to some tissues (e.g. skeletal muscle) but not
to others (e.g. brain). Lysates from Icmt
/
embryos lacked the ability to methylate either recombinant K-Ras or
small molecule substrates (e.g.
N-acetyl-S-geranylgeranyl-L-cysteine). In addition, Icmt
/
cells lacked the ability to
methylate Rab proteins. Thus, Icmt appears to be the only enzyme
participating in the carboxyl methylation of isoprenylated proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) were identified on Southern blots with a
5'-flanking probe (10). Two clones, each with a single neo
integration, were used to produce Icmt+/
mice. Timed
matings of Icmt+/
mice were performed to assess the
viability of homozygotes (Icmt
/
) at different stages of
development. The genotype of each embryo was determined by Southern
blot analysis (10). Icmt
/
fibroblasts were produced from
mouse embryos as previously described (8).
/
ES Cells--
To
assess the contribution of Icmt-deficient ES cells to
different tissues, two independent lines of Icmt
/
ES
cells (10) were injected into C57BL/6 blastocysts. Ten male
chimeric mice were obtained; all were 35-75% chimeras as judged by
coat color. At 8 weeks of age, the mice were sacrificed, and genomic
DNA was purified from multiple tissues and analyzed by Southern blot
with a 32P-labeled Icmt probe. The ratio of
Icmt
to Icmt+ bands in each tissue was
determined by phosphorimager.
- and
-subunits and then purified by cation exchange
chromatography followed by gel filtration chromatography. REP was also
expressed in Sf9 cells as a C-terminal histidine-tagged fusion
protein and was purified by Ni2+-Sepharose affinity chromatography.
80 °C.
/
cells--
To assess the level of methylation substrates within cells,
whole-cell lysates (Icmt+/+, Icmt+/
, and
Icmt
/
) were incubated with
S-adenosyl-L-[methyl-14C]methionine
and recombinant Ste14p (10 µg of membrane protein from Sf9
cells that overexpress yeast STE14) (7). The amount of
base-labile methylation was quantified as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mice were produced from two
independent lines of ES cells. Genotyping of 21-day-old offspring from
Icmt+/
intercrosses revealed that about two-thirds (58 of
82) were heterozygotes, and the remainder were wild-type. Genotyping of
embryos revealed that Icmt
/
embryos constituted 25% of
the litter until embryonic day 10.5 (E10.5, Fig.
1B). By E11.5, there were only
a few viable Icmt
/
embryos; those embryos had beating
hearts and red blood cells but were far smaller than heterozygous and
wild-type mice (Fig. 1C). Virtually all of the
Icmt
/
mice died by E12.5, several days before the first
of the homozygous Rce1 knockout embryos started to die (8).
Histologic studies of Icmt
/
embryos did not reveal a
specific cause of death.
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Fig. 1.
Death of Icmt /
embryos
at mid-gestation. A, Southern blot of the genomic DNA
from the yolk sacs of Icmt+/+, Icmt+/
, and
Icmt
/
embryos. Genomic DNA was digested with
BamHI. The Southern blot was hybridized with a 5'
probe (10). The BamHI fragment in the wild-type allele is
5.0 kb, whereas it is 6.8 kb in the targeted allele.
B, percentages of Icmt+/+ (
),
Icmt+/
(
), and Icmt
/
(
) embryos
surviving at different time points. C, Icmt+/+,
Icmt+/
, and Icmt
/
embryos at E11.5.
/
ES
cells (10) and performed Southern blots to assess the relative
capacities of Icmt+/+ and Icmt
/
cells to
populate different tissues (Fig.
2A). The Icmt
/
cells contributed significantly to the development of skeletal muscle, as shown by the 1:1 ratio of Icmt
and Icmt+
band intensities in the genomic DNA of that tissue but made a
negligible contribution to the formation of the brain. The
Icmt
band was virtually undetectable in brain, and the
Icmt+/Icmt
band ratio was about 8:1. High
Icmt+/Icmt
ratios were also present in liver
and testis. Interestingly, the extent to which Icmt
/
cells contributed to the formation of each tissue appeared to be
inversely correlated with the normal levels of Icmt
expression in that tissue. Thus, as documented by a Northern blot (Fig.
2B), Icmt expression in wild-type mice was quite
low in skeletal muscle, but high in the brain and liver. Measurements
of enzyme activity in wild-type mice were in general agreement with the
Northern blot results, with high activity levels in the brain, testis,
and liver and low levels in skeletal muscle (Fig. 2C).
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Fig. 2.
Analyzing Icmt expression in
different tissues. A, ratio of
Icmt+/Icmt alleles in the organs of chimeric
mice (n = 10) generated with Icmt
/
ES
cells. The DNA was prepared from multiple tissues, digested with
BamHI, and analyzed on Southern blots with an
Icmt probe (10). The ratio of the 5.0-kb Icmt+
band to the 6.8-kb Icmt
band for each organ (mean ± S.D.) is shown. B, Icmt expression in
Icmt+/+ tissues assessed with a CLONTECH mouse
multiple tissue Northern blot. C, ability of lysates from
tissues of Icmt+/+ mice to methylate
N-acetyl-S-geranylgeranyl-L-cysteine.
Methylation (pmol/mg cell protein/min) was measured with a
base-hydrolysis assay (10). Bars show mean ± S.D.
/
embryos retained the capacity
to methylate isoprenylated proteins, perhaps through a redundant enzymatic activity, we tested the capacity of lysates of
Icmt
/
embryos to methylate farnesyl-K-Ras (Fig.
3A). No enzymatic activity above background levels was identified. We considered the possibility that a redundant enzymatic activity might not be able to methylate K-Ras and therefore tested the ability of Icmt
/
lysates
to methylate two small molecule substrates,
N-acetyl-S-geranylgeranyl-L-cysteine and
N-acetyl-S-farnesyl-L-cysteine
(Fig. 3B). The results of those studies were identical to
the results of the K-Ras experiments, loss of activity in lysates of
Icmt
/
embryos. We also measured methyltransferase
activity against small molecule substrates in lysates from
Icmt+/
embryos and tissues from adult Icmt+/
mice (liver, brain, and heart). Activities were invariably reduced by
50% (data not shown), not less than 50%, as would be the case if
there were redundant methyltransferase activities.
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Fig. 3.
Methyltransferase activity in embryo
lysates. A, ability of lysates from Icmt+/+,
Icmt+/ , and Icmt
/
embryos to methylate
farnesyl-K-Ras4B. B, methylation of
N-acetyl-S-geranylgeranyl-L-cysteine
(AGGC) and
N-acetyl-S-farnesyl-L-cysteine
(AFC) by embryo lysates. Bars show the mean ± S.D. In some cases, the S.D. were low so that the error bar is not
visible above the column. C, accumulation of methylation
substrates in embryo lysates. Lysates (100 µg) from
Icmt+/+, Icmt+/
, and Icmt
/
embryos (n = 2) were mixed with
S-adenosyl-L-[methyl-14C]methionine
(10 µM) and yeast Ste14p. Methylation was assessed with a
base hydrolysis assay. The bar graph shows the average of
values for the two embryos of each genotype.
/
embryos
(i.e. an accumulation of cellular proteins that could be
methylated by the yeast ortholog of Icmt, Ste14p, Fig.
3C).
/
embryos would retain the
ability to methylate CXC Rab proteins. This was not the
case. Lysates from Icmt
/
cells were incapable of
methylating three different CXC Rab proteins, although
those proteins were readily methylated by lysates from
Icmt+/+ cells (Fig. 4). The
importance of Rab methylation by Icmt remains obscure, but one obvious
possibility is that it is important for membrane targeting. In
preliminary experiments, we have used cell fractionation experiments
and the expression of GFP·Rab fusions to assess the localization of
Rab6 (a Golgi CXC Rab protein) in Icmt
/
and Icmt+/+ cells but did not observe noticeable differences
(data not shown).
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Fig. 4.
Methylation of CXC Rab
proteins by lysates from Icmt+/+ and
Icmt /
cells. Recombinant Rab3B, Rab3D, and
Rab6 were geranylgeranylated and tested as methylation substrates with
lysates from Icmt+/+ and Icmt
/
fibroblasts. Bars show the mean ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
embryos dying between E10.5 and E11.5. Importantly, our biochemical studies with embryo lysates did not uncover a residual or redundant Icmt-like activity, and
the lysates manifested a striking increase in Ste14p substrates.
/
embryos probably died because
Icmt-deficient cells failed to grow and contribute to the
formation of various organs. Southern blots of tissues from chimeric
mice generated with Icmt
/
cells revealed that
Icmt-deficient cells are severely defective in their
capacity to contribute to the formation of certain organs (e.g. liver and brain) although they retained the ability to
contribute to the formation of others (e.g. skeletal
muscle).2 We doubt that this
finding was spurious, for several reasons. First, similar results were
obtained with two lines of Icmt
/
ES cells. Second, there
was a reasonably strong inverse correlation between normal levels of
Icmt expression and the ability of Icmt
/
ES
cells to contribute to the formation of a tissue. Third, the Icmt chimeric mouse experiments were performed in parallel
with studies with Zmpste24
/
ES
cells,3 which robustly
populated all of the tissues of chimeric
mice.4
/
embryos than in Rce1
/
embryos? One
possibility is simply that Icmt has more substrates than Rce1. Indeed,
our experiments revealed for the first time that the CXC Rab
proteins (which are not processed by Rce1) are methylated by
Icmt. Rab proteins terminating in CXC and CC are
geranylgeranylated at both cysteines (12). The CXC Rab
proteins, but not the CC Rab proteins, are then carboxyl-methylated (12, 19-23). For example, Rab3a and Rab4, which terminate in Cys-Ala-Cys and Cys-Gly-Cys, respectively, are carboxyl methylated (19,
20), whereas Rab1A and Rab2, which terminate in Cys-Cys, are not (12,
23). Interestingly, replacement of the CXC terminus of Rab3a
with CC abolishes methylation, whereas the opposite result is obtained
when the CC terminus of Rab1A is replaced with a CXC sequence (12).
/
embryos is that the positioning of the carboxylate anion on CAAX proteins has a profound
influence on the binding of isoprenylated proteins to membranes,
protein partners, or both. Both Rce1 and Icmt
deficiency eliminate the carboxyl methylation of CAAX
proteins and thereby leave the protein with a C-terminal carboxylate
anion rather than an
-methyl ester. However, the position of the
carboxylate anion differs, being within the isoprenylcysteine residue
in the setting of Icmt deficiency and located three amino
acids downstream in the setting of Rce1 deficiency.
Methylation itself is clearly important for membrane binding:
methylating
N-acetyl-S-farnesyl-L-cysteine
increases the partitioning of that molecule into the organic phase of
an n-octanol/water mixture (24), and methylating
farnesylated CAAX peptides increases their binding to
synthetic liposomes (25). The impact of carboxylate anion positioning
(i.e. on the prenylcysteine or on the end of the C-terminal
tripeptide) on lipid-binding properties has not been studied in a
similar fashion. However, it is easy to imagine that a more vicinal
carboxylate anion (i.e. with Icmt deficiency)
might be more potent in inhibiting the association of the isoprenyl
lipid with membranes.
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ACKNOWLEDGEMENTS |
---|
We thank Viktoria Gustafsson for technical assistance, Matthew Ashby and Steven Clarke for helpful discussions, and Stephen Ordway for criticisms of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL41633 and AG15451 (to S. G. Y.) and GM46372 (to P. J. C), a Wellcome Trust Programme Grant (to M. C. S.), and grant awards from the University of California Tobacco-related Disease Research Program (to M. O. B. and S. G. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Gladstone Institute of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-695-3774; Fax: 415-285-5632; E-mail: mbergo@gladstone.ucsf.edu.
§§ Recipient of a Ph.D. Student Award from Fundação Ciência e Tecnologia of Portugal.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.C000831200
2 Although no Southern blots were performed on skin DNA, the percentage of brown fur on the chimeric animals suggested that Icmt-deficient ES cells are fairly robust in contributing to the formation of the hair follicles.
3 Zmpste24 is the mouse ortholog of AFC1 (STE24) in Saccharomyces cerevisiae (2, 27, 28).
4 G. Leung, M. Bergo, and S. Young, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: CAAX, C-terminal motif consisting of Cys followed by two aliphatic residues (A) and any amino acid (X); Icmt, isoprenylcysteine carboxyl methyltransferase; ES, embryonic stem; AGGC, N-acetyl-S-geranylgeranyl-L-cysteine; AFC, N-acetyl-S-farnesyl-L-cysteine; GGPP, geranylgeranylpyrophosphate; REP, Rab escort protein; RabGGTase, Rab geranylgeranyltransferase; kb, kilobases.
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REFERENCES |
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