 |
INTRODUCTION |
Post-translational modifications are essential for the membrane
targeting of Ras proteins and many other proteins containing a
"CAAX sequence" at their C terminus (1-4).
CAAX proteins terminate with the amino acids
-C-A-A-X, where the "C" is a cysteine, the two "A" residues are usually aliphatic amino acids, and
the "X" can be one of several amino acids.
CAAX proteins undergo three sequential enzymatic
post-translational modifications. First, either a farnesyl or
geranylgeranyl isoprenoid lipid is attached to the thiol group of the
cysteine residue by cytosolic protein prenyltransferases (5). In
general, the cysteine is geranylgeranylated when the X
residue is a leucine or a phenylalanine; otherwise, the protein is
farnesylated (6, 7). Second, the last three amino acids of the protein
(i.e. the -AAX) are removed by a specific endoprotease; this step is prenylation-dependent and is
thought to take place on the cytoplasmic surface of the endoplasmic
reticulum (8). Finally, the C group of the prenylcysteine is methylated by an endoplasmic reticulum-associated methyltransferase (1, 9, 10). In
addition to these three processing events, some CAAX
proteins, including Ha-Ras, Ki-Ras4A, and N-Ras, can be further modified by palmitoylation of upstream cysteine residues (11).
The mammalian prenyltransferase enzymes responsible for farnesylation
and geranylgeranylation of the CAAX proteins have been defined (2, 5, 12), and a mammalian enzyme responsible for methylating
the prenylcysteine of CAAX proteins was identified recently
(13). However, progress in defining the mammalian enzymes for the
middle step of the triad, the endoproteolysis step, has been limited.
Enzymatic activities capable of carrying out the endoproteolysis step
have long been recognized (10, 14-18). However, only very recently
have specific molecules been linked to that step, and virtually all of
this progress has occurred in yeast. Two genes from Saccharomyces
cerevisiae, RCE1 and AFC1, have been shown
to be involved in the proteolytic processing of several yeast
CAAX proteins (19, 20). Rce1p (for Ras and
a-factor-converting enzyme) is essential for cleaving the
C-terminal three amino acids of a yeast Ras protein, Ras2p, and is
active in processing the yeast mating pheromone a-factor
(19, 20). Afc1p (for a-factor-converting enzyme, also
described as Ste24p; Ref. 21) carries out two successive proteolytic
processing steps for the precursor to a-factor: cleavage of
the C-terminal three amino acids from the farnesylated precursor and
the subsequent cleavage of 7 amino acids from the N-terminal portion of
the protein (20-22). Afc1p does not process yeast Ras2p (19), nor does
it process human farnesyl-Ki-Ras in an in vitro assay
(8).
For several years, it has been clear that the prenylation of
CAAX proteins, particularly the Ras proteins, is critical
for plasma membrane targeting and for biological function (11, 23). However, the functional importance of the proteolysis and methylation steps has been less obvious. Those "post-prenylation" steps may simply render prenylated proteins more hydrophobic and thereby enhance
their membrane affinity (24, 25). It has also been suggested that those
processing steps could enhance the stability of some prenylated
proteins (26). In any case, several studies have suggested that the
post-prenylation processing steps might be important for the function
of the Ras proteins. For example, a mutant Ki-Ras4B that was prenylated
but not further processed exhibited reduced transforming activity when
expressed in 3T3 fibroblasts (27). Moreover, in
RCE1-deficient yeast (rce1
), Ras2p was
mislocalized to the cytosol and internal membranes, and the yeast were
protected from the heat sensitivity elicited by the expression of a
mutationally activated Ras2p. Interestingly, rce1
yeast
grew as well as wild-type cells under normal conditions, despite the
defect in Ras processing (19). The fact that blocking Rce1p-mediated
CAAX protein proteolysis appeared to attenuate the
biological effects of an activated yeast Ras protein without affecting
the growth of yeast under normal conditions has led to speculation that
inhibiting the proteolysis step might be a promising therapeutic
strategy for treating human cancers caused by mutationally activated
Ras proteins (19, 28).
The purpose of this study was to define the mammalian enzyme
responsible for Ras endoproteolytic processing and to gain insights into the physiologic consequences of blocking that processing step. At
the time that the RCE1 gene was identified in yeast, expressed sequence tags
(ESTs)1 containing sequence
homology to that gene existed within the mouse EST data bases. To
determine whether these ESTs represented a bona fide functional
homologue of the yeast gene with a role in the processing of mammalian
Ras proteins, the mouse ESTs were used to identify a large genomic
clone spanning the mouse gene (designated Rce1), which was
then disrupted by gene targeting in mouse embryonic stem (ES) cells.
Here, we describe the phenotype of Rce1-deficient mice and
provide evidence that Rce1 is essential for the proteolytic
processing of the mammalian Ras proteins. We also show that
Rce1-deficient cells are defective in the proteolytic cleavage of other prenylated proteins, establishing a broader role for
the product of the Rce1 gene in the processing of
CAAX proteins.
 |
EXPERIMENTAL PROCEDURES |
Identification of Mouse Rce1 ESTs and a Mouse Rce1 Genomic
Clone--
An XREFdb query of GenBankTM sequences with the
yeast RCE1 coding sequences identified two mouse EST
matches: I.M.A.G.E. clones 331228 (0.8 kb) and 608689 (1.1 kb)
(Research Genetics, Huntsville, AL). These ESTs shared 71% amino acid
homology and 52% identity with several portions of the yeast
RCE1 gene in a region spanning amino acids 147-257. DNA
sequencing and restriction mapping revealed that the mouse ESTs
contained overlapping sequences spanning the majority of the mouse
Rce1 cDNA. Screening of a bacterial artificial chromosome library of strain 129/Sv mouse genomic DNA (Research Genetics) with the 1.1-kb EST identified a clone (427 J 4) that spanned
the mouse Rce1 gene.
Analysis of the Tissue Distribution of Rce1 Gene
Expression--
An RNA Master Blot (CLONTECH, Palo
Alto, CA) was used to identify the tissues that expressed the
Rce1 gene. The blot was probed with a 223-base pair
Rce1 cDNA fragment (corresponding to amino acids
136-210), which was amplified from a mouse liver cDNA library (CLONTECH, Palo Alto, CA) with oligonucleotide
primers 5'-TTGCCCTTGTGACCTGACAGATGG-3' and
5'-GAGTGGGCAGGTGAACACAGCAGG-3'. To assess the relative levels of
Rce1 gene expression in different tissues, the blot was
analyzed with a phosphorimager (Fuji Bio-Imaging Analyzer, BAS 1000 with MacBAS; Fuji Medical Systems, Stamford, CT).
Generation of Rce1-deficient Mice--
A sequence replacement
gene-targeting vector was constructed from a 10.5-kb BamHI
fragment of the bacterial artificial chromosome that spanned the
Rce1 gene. The vector was constructed in the plasmid
pKSloxPNT (29) (provided by Dr. A. Joyner), which contains polylinker
restriction sites, a thymidine kinase gene (tk), and a
neomycin resistance gene (neo) flanked by loxP
sites. The 5' arm of the vector, 3.8 kb long, contained the promoter
sequences of the Rce1 gene and all of the coding sequences
located 5' to an Eco47III site within an exon of the
Rce1 gene (corresponding to amino acid 115 of the protein);
the 3' arm (on the 3' side of the neo) extended from an
EcoRV site located within the last exon of the gene (within
the 3'-untranslated sequences of the mRNA) to an EcoRI
site located ~3.6 kb 3' to the gene. Thus, the neo
replaced the Eco47III-EcoRV segment of the
Rce1 gene, which codes for the C-terminal two-thirds of the
protein (amino acids 115-329). This vector was electroporated into
mouse ES cells, and targeted colonies were identified by Southern
analysis of XbaI-cleaved genomic DNA with a 1-kb
EcoRI-BamHI probe located 3' to the sequences in
the gene-targeting vector. Two targeted ES cell clones were used to
generate Rce1-deficient mice, according to published
techniques (30). All mice described here had a mixed genetic background
(~50% C57BL/6 and ~50% 129/Sv). The mice were weaned at 21 days
of age, housed in a barrier facility with a 12-h light/dark cycle, and
fed a chow diet containing 4.5% fat (Ralston Purina, St. Louis, MO).
Examination of Rce1-deficient Mouse Embryos--
Timed matings
were established with Rce1+/
males and females
as described previously (31). Pregnant female mice were euthanized between 10.5 and 20.5 days post coitus. After dissection of the embryo
and fetal membranes from the uterus, embryos were fixed in 3%
paraformaldehyde, and fetal membranes were used for genotyping. The
embryos were sectioned, stained with hematoxylin and eosin, and
examined by light microscopy.
Preparation of Fibroblasts from Rce1-deficient Mouse
Embryos--
To isolate primary embryonic fibroblasts,
Rce1+/
intercrosses were established, and
pregnant females were sacrificed at 11.5 days post coitus. Fetal
membranes were used for genotyping. The embryos were incubated in 5.0 ml of 0.25% trypsin-EDTA (Life Technologies, Inc.) for 8 h at
4 °C, followed by a 20-min incubation at 37 °C. After removal
from the trypsin solution, the embryos were mechanically disrupted by
repeated pipetting in 5 ml of Dulbecco's modified Eagle's medium
supplemented with 10% v/v fetal bovine serum, L-glutamine, nonessential amino acids, penicillin-streptomycin (all from Life Technologies), and 2-mercaptoethanol (Sigma; final concentration, 100 µM). The debris was allowed to settle, and the cell
suspension was removed, diluted to 25 ml with medium, and plated in
T150 flasks. Approximately 1 × 107 cells were
obtained from each embryo. The cells were grown in an incubator with
7% CO2 at 37 °C. To immortalize the fibroblasts, cells
were passaged according to a 3T6 protocol (32), in which cells are
harvested and counted every 3 days and replated at a density of 2 × 106 cells/100-mm dish.
Western Blot Analysis of Ras Proteins in Rce1-deficient
Mice--
To prepare cell lysates of primary embryonic fibroblasts,
confluent cells were harvested from a 100-mm plate in 0.5 ml of RIPA
lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 5 mM MgCl2, 1% Triton X-100, 0.5%
sodium deoxycholate, 0.1% SDS, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and disrupted by sonication for 10 s in a Branson model 400 sonifier (duty cycle constant, output control 0.3; Danbury, CT). To obtain whole embryo lysates, E14.5 mouse embryos were isolated from pregnant
Rce1+/
females that had been mated with a
Rce1+/
male. The embryos were homogenized in
3.0 ml of phosphate-buffered saline (PBS; Digene Diagnostics,
Beltsville, MD) containing 1% SDS and 50 mM
2-mercaptoethanol for 15 s with a Kinematica Polytron (Brinkman
Instruments, Palo Alto, CA). The samples were then heated to 95 °C
for 5 min and loaded onto an SDS/10-20% gradient polyacrylamide gel.
After electrophoretic transfer of the proteins to a nitrocellulose membrane, Western blots performed with the "pan-Ras" monoclonal antibody Ab-4 (i.e. binds to N-Ras, Ki-Ras, and Ha-Ras;
Oncogene Science, Uniondale, NY) or the Ki-Ras-specific monoclonal
antibody Ab-1 (Oncogene Science), followed by an incubation with
horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham
Pharmacia Biotech). Antibody binding was detected with the Enhanced
Chemiluminescence kit (Amersham Pharmacia Biotech).
To immunoprecipitate the Ras proteins from fibroblasts or from the
tissues of mouse embryos, lysates (200 µg of embryo lysate protein or
the complete lysate from a 100-mm plate of confluent fibroblasts) were
adjusted to 1.0 ml with RIPA buffer and pre-cleared by an incubation
with protein A-Sepharose CL-4B beads (Sigma) or protein G-agarose beads
(Boeringher Mannheim) for 30 min at 4 °C. The supernatant was then
incubated with 5 µg of Y13-259 (Santa Cruz Biotechnology, Santa
Cruz, CA), a pan-Ras-specific rat monoclonal antibody overnight at
4 °C. The immune complexes were then incubated with protein A- or
protein G-agarose beads for 2 h at 4 °C and centrifuged for 5 min at 12,500 × g. After three washes with RIPA
buffer, the pellets were resuspended in 20 µl of sample buffer.
Immunoprecipitates were size-fractionated on an SDS/10-20% gradient
polyacrylamide gel, and Western blots were performed with either
antibody Ab-4 or Ab-1.
Methylation of Ras Proteins in Rce1
/
Fibroblasts--
To determine whether the C terminus of Ras was
methylated in Rce1+/+ and
Rce1
/
embryonic fibroblasts, we used a
modification of the base-labile methylation assay described by Clarke
et al. (1). Briefly, 100-mm dishes containing approximately
2.5 × 106 fibroblasts were incubated in the presence
of 1 mCi of
L-[methyl-3H]methionine (80 Ci/mmol; Amersham Pharmacia Biotech) and [35S]cysteine
(>1000 Ci/mmol; Amersham Pharmacia Biotech) in methionine- and
cysteine-free Dulbecco's modified Eagle's medium/10% fetal bovine
serum in a 7% CO2 incubator for 24 h at 37 °C. The
medium was removed, and the cells were washed with 5 ml of ice-cold
PBS, scraped from the dish with a rubber policeman into 1 ml of
ice-cold PBS, and centrifuged at 500 × g. Cell pellets
were resuspended in 1.0 ml of RIPA lysis buffer and disrupted by
sonification, as described above. Ras proteins were then
immunoprecipitated with antibody Y13-259, and the immunoprecipitate
was size-fractionated on an SDS/10-20% gradient polyacrylamide gel.
The gel was fixed in isopropanol, water, and acetic acid (25:65:10,
v/v), soaked for 30 min in Amplify (Amersham Pharmacia Biotech), and
dried in a vacuum oven at 80 °C. The dried gel was imaged with a
phosphorimager, and the area corresponding to the Ras band was excised
and used to quantify the amount of [3H]methanol released
by base hydrolysis of the cysteine methyl esters. The gel slice was
placed in a 1.5-ml capless microcentrifuge tube, mixed with 200 µl of
2 M NaOH, and lowered into 20-ml scintillation vial
containing 6 ml of Safety-Solve II counting fluor (RPI) fluid. The vial
was then capped and incubated at 55 °C; within the capped vial, the
[3H]methanol released from the cysteine methyl esters
diffuse into the fluor, whereas the labeled protein remains in the gel
slice. After 24 h, the microcentrifuge tubes were removed, and the
radioactivity in the vials was counted.
CAAX Endoprotease Assay--
CAAX endoprotease
activities were measured using a coupled assay. Recombinant prenyl
proteins were first used as substrates in a proteolysis assay in which
the protease source was membranes from either
Rce1+/+ or Rce1
/
fibroblasts. The degree of specific proteolytic processing of the
prenyl proteins was then determined by quantifying their ability to be
methylated by the yeast prenyl protein carboxymethyltransferase STE14
produced in Sf9 cells.
Substrate proteins containing a CAAX sequence were expressed
in Escherichia coli, prenylated in vitro with
recombinant protein farnesyltransferase or protein
geranylgeranyltransferase, and either purified (in the case of
farnesyl-Ki-Ras and geranylgeranyl-Ki-Ras) or simply concentrated as a
mixture of prenylated protein, nonprenylated protein, and the
prenyltransferase (33). Ki-Ras, N-Ras, and Rap-1B were modified at the
N terminus with a hexahistidine tag, whereas G
1 and
Ha-Ras were unmodified. To prepare membranes from
Rce1+/+ and Rce1
/
embryonic fibroblasts, cells were lysed by sonication. After centrifugation at 500 × g for 10 min to remove
cellular debris and nuclei, the membrane fraction was ultracentrifuged
at 200,000 × g for 1.5 h and resuspended in 50 mM Tris-HCl at protein concentrations of 10-25 mg/ml.
To assay for the prenyl protein-specific protease, prenyl protein
substrates and membrane fractions were mixed in 50 µl of a 100 mM Hepes buffer, pH 7.5, containing 5 mM
MgCl2. After a 30-min incubation at 37 °C, the
proteolysis reactions were stopped, and the methyltransferase reactions
were initiated by adding a mixture (20 µl) containing 5 mM NaHPO4, 87.5 mM EDTA, a variety of protease inhibitors (300 µM
N-tosyl-L-phenylalanine chloromethylketone, 1 mM phenylmethylsulfonyl fluoride, 10 mM
1,10-phenanthroline), 20 µg of Sf9/STE14 membranes,
and 17.5 µM
[3H]S-adenosylmethionine (1.5 Ci/mmol). After
a 20-min incubation at 37 °C, the methylation reaction was
terminated by adding 0.5 ml of 4% SDS. Bovine brain cytosol (50 µg
of protein) was added as a carrier protein, and the mixture was
incubated for 20 min at room temperature, whereupon the proteins were
precipitated by adding 0.5 ml of 30% trichloroacetic acid.
Precipitated proteins were collected on glass fiber filters (for the
Ras proteins and Rap1B) or nitrocellulose filters (for
G
1), and the extent of proteolysis was determined by
quantifying 3H incorporation by scintillation counting.
Subcellular Fractionation and Localization of Ras Proteins in
Fibroblasts--
To determine the degree of membrane association of
Ras, subcellular fractionation of primary cells and transformed cells
was performed as described (34). For each sample, 100-mm dishes containing confluent fibroblasts were washed with ice-cold PBS, and
cells were collected in 1.0 ml of PBS and centrifuged at 500 × g for 10 min. Cell pellets were swelled with 1225 µl of
hypotonic buffer (10 mM Tris-HCl, pH 7.5, 1.0 mM MgCl2, 0.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin, and 1 µM dithiothreitol) and placed on ice for
10 min. Cells were disrupted with an ice-cold Dounce tissue
homogenizer, after which 225 µl of 1 M NaCl was added. A
total of 450 µl of this solution ("total lysate") was transferred
to a microfuge tube and set aside. The remaining 1000 µl was
transferred to a polycarbonate ultracentrifuge tube and spun at
100,000 × g for 30 min at 4 °C. The supernatant fluid (S100, representing the cytosolic fraction) was transferred to a
new microcentrifuge tube; the pellet (P100, representing the membrane
fraction) was resuspended in 850 µl of hypotonic buffer and 150 µl
of 1 M NaCl. Next, 50 µl of 10 × Hi-SDS RIPA buffer
was added to total lysate sample, and 110 µl was added to the S100
and P100 fractions. After incubation on ice for 10 min, the lysates
were clarified by centrifugation at 25,000 × g for 30 min at
4 °C. Supernatants were transferred to a new microfuge tube, and
immunoprecipitations, gel fractionations, and Western blot detection of
the Ras proteins were performed as described earlier.
To further assess the association of the Ras proteins with the mbranes
of Rce1
/
and Rce1+/+
embryonic fibroblasts, we prepared an enhanced green fluorescent protein-Ki-Ras fusion protein in which the last 18 amino acids corresponded to the mouse Ki-Ras sequence. Complementary
oligonucleotides corresponding to the last 18 amino acids of mouse
Ki-Ras
(5'-AATTCTGGTAAAAAGAAGAAAAAGAAGTCAAAGACAAAGTGTGTAATTATGTAGA-3' and 5'-GATCTCTACATAATTACACACTTTGTCTTTGACTTCTTTTTCTTCTTTTTACCAG-3') were
annealed and ligated into the vector pEGFP-C1
(CLONTECH, Palo Alto, CA), which had been cleaved
with EcoRI and BamHI. The plasmids were then
sequenced to verify that the sequence of the oligonucleotide was in
frame with that of the green fluorescent protein.
Rce1
/
and Rce1+/+
embryonic fibroblasts were transfected with either 2.0 µg of the the
parental green fluorescent protein plasmid (EGFP)-Ki-Ras construct or
the EGFP with SuperFect Reagent (Qiagen, Valencia, CA), according to
the manufacturer's recommendations. Cells were fixed in 4% formalin
24-28 h after transfection, mounted, and viewed by confocal microscopy.
Adoptive Transfer of Hematopoietic
Cells--
Rce1+/
mice were intercrossed to
produce Rce1
/
,
Rce1+/
, and Rce1+/+
embryos. Pregnant females were sacrificed on day 14.5-15.5 post coitus, and the embryos were removed. Fetal livers were transferred to
1 ml of Iscove's modified Dulbecco's medium supplemented with 20%
fetal calf serum. Single-cell suspensions were prepared by drawing the
livers through progressively smaller needles (a 22-gauge needle × 2 followed by a 25-gauge needle × 2). DNA was extracted from the
remaining embryonic tissues, and a polymerase chain reaction-based assay was used to determine Rce1 genotypes before adoptive
transfer. A total of 2-10 × 106 mononuclear cells
were injected into the tail veins of recipients previously subjected to
1000 centigrays of total body irradiation. This irradiation protocol
destroys recipient hematopoiesis and is associated with >95% survival
of animals engrafted with wild-type fetal liver cells. Complete blood
counts were performed monthly for at least 4 months after adoptive
transfer. The genotype of circulating blood cells was assessed by
Southern blot analysis.
To assess the growth of colonies derived from colony-forming unit
granulocyte-macrophage progenitors, mononuclear cells from fetal livers
were plated on the same day that they were harvested in duplicate 35-mm
plates (5 × 104 cells/plate) in fully supplemented
Iscove's modified Dulbecco's medium (Stem Cell Technologies,
Vancouver, Canada) medium containing 0.8% methylcellulose, 30% fetal
calf serum, and L-glutamine. The fetal liver cells and
recombinant murine granulocyte-macrophage colony-stimulating factor
were added directly to the methylcellulose-containing medium, and the
solution was mixed thoroughly before plating. The cells were grown at
37 °C in a humidified 5% CO2 incubator. Colonies
derived from granulocyte-macrophage colony forming units were counted
by indirect microscopy.
 |
RESULTS |
Tissue Pattern of Rce1 Gene Expression--
To assess the pattern
of Rce1 gene expression in mice, an RNA dot blot was probed
with a mouse Rce1 cDNA clone. Rce1 expression was observed in all tissues examined but was particularly high in the
heart (Fig. 1). Of note, Rce1
expression was detectable in early stage (E7.0) mouse embryos.

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Fig. 1.
Phosphorimager analysis of an RNA dot blot,
illustrating relative levels of Rce1 gene expression
in different mouse tissues. A 223-base pair Rce1
cDNA probe was hybridized to a multiple tissue mRNA dot blot.
The amount of RNA loaded onto each dot was normalized to the mRNA
expression levels of eight housekeeping genes.
|
|
Generation of Rce1 Knockout Mice--
Southern blots of
EcoRI-, BamHI-, or SacI-cleaved mouse
genomic DNA probed with the 1.1-kb EST revealed distinct, single bands (EcoRI, 8.0 kb; BamHI, 10 kb; SacI, 4 kb), suggesting that the mouse genome contains only a single copy of
the Rce1 gene (not shown). To inactivate the Rce1
gene, we constructed a sequence replacement gene-targeting vector
designed to delete most of the coding sequences of the Rce1
gene (Fig. 2A). After
electroporation of the vector into mouse ES cells, Southern blot
analysis of 196 drug-resistant ES cell clones revealed that eight were
correctly targeted. Two of these clones were used to generate high
percentage male chimeric mice, which were bred with C57BL/6 females.
Each of the chimeras transmitted the targeted Rce1 mutation
to their progeny, generating heterozygous Rce1 knockout mice
(Rce1+/
). Male and female
Rce1+/
mice were healthy and fertile and were
intercrossed to generate homozygotes. From 25 litters, we
identified 53 wild-type mice (Rce1+/+),
100 Rce1+/
mice, and 4 knockout
homozygotes (Rce1
/
), indicating that
homozygosity for the Rce1 mutation caused significant embryonic lethality. All four live-born
Rce1
/
mice died, two perinatally and two at
10 days of age. The two mice that survived 10 days were severely runted
but otherwise appeared normal. A visual inspection of the major organ
systems in one of the Rce1
/
mice revealed no
obvious cause of death. The phenotypes of Rce1-deficient mice derived from the two different targeted ES cell clones were identical.

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Fig. 2.
Generation of Rce1-deficient
mice. A, a sequence replacement strategy to knock out
the Rce1 gene. The assessment of gene-targeting events in ES
cells and mouse genotyping were performed by Southern blot analysis,
using the indicated probe located 3' to sequences in the gene-targeting
vector. B, Southern blot identification of
Rce1+/+, Rce1+/ , and
Rce1 / embryos with XbaI-cleaved
mouse genomic DNA and the 3'-flanking probe. Also shown is a Southern
blot of XbaI-cleaved mouse genomic DNA, using a
"deletion" probe spanning the Eco47III-EcoRV
fragment of the Rce1 gene that was deleted by the gene-targeting
event.
|
|
Fig. 2B illustrates the Southern blot identification of
Rce1+/
and Rce1
/
mice and also shows a Southern blot with a probe that spanned the
segment of the Rce1 gene that was deleted by the knockout mutation. As expected, that probe did not hybridize to any DNA fragment
in the Rce1
/
mice.
Rce1 Deficiency Is Lethal in Mice--
To determine the timing of
the demise of Rce1
/
embryos, we established
26 timed pregnancies from intercrosses of
Rce1+/
mice. Prior to E15.5,
Rce1
/
and Rce1+/+
embryos were present in the expected numbers (~25% of the total); however, the percentage of Rce1
/
embryos
declined sharply after E15.5 (Fig. 3).
Gross inspection of the viable Rce1
/
,
Rce1+/+, and Rce1+/
embryos after E15.5 revealed no consistent differences in morphology, organogenesis, stage of development, color, or size. Histological examination of 12.5-, 15.5-, 16.5-, 17.5-, and 18.5-day wild-type (n = 10) and knockout (n = 10) embryos
revealed no apparent defect in the development of major organ systems,
including the brain, intestinal tract, heart, lungs, kidneys, and
placenta. The hearts and thoracic cavities of representative
Rce1
/
and Rce1+/+
embryos are shown in Fig. 4
(A-D). High power micrographs of embryonic livers
revealed abundant hepatocytes and hematopoietic cells in both
Rce1
/
and Rce1+/+
embryos (Fig. 4, E and F).

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Fig. 3.
Percentages of
Rce1+/+ and
Rce1 / embryos in litters from
intercrosses of Rce1+/ mice at different
times during gestation.
|
|

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Fig. 4.
Hematoxylin/eosin-stained sections of tissues
from Rce1 / and
Rce1+/+ embryos. A, saggital
section of the left lateral thoracic cavity from an E15.5
Rce1 / embryo. B, saggital section
of the left lateral thoracic cavity from an E15.5
Rce1+/+ embryo. C, saggital section
of the mid-thoracic cavity from an E15.5
Rce1 / embryo. D, saggital
section of the mid-thoracic cavity from an E15.5
Rce1+/+ embryo. E, liver from an
E15.5 Rce1 / embryo. F, liver from
an E15.5 Rce1+/+ embryo.
|
|
Because the Rce1
/
embryos displayed no overt
defect in organogenesis and survived until late in gestation, we
suspected that it would be possible to culture embryonic fibroblasts
from Rce1
/
embryos. This suspicion was
confirmed; the yield and morphology of primary embryonic fibroblasts
from E11.5 Rce1
/
and
Rce1+/+ embryos were identical. Also, the growth
rate of Rce1
/
and
Rce1+/+ fibroblasts were indistinguishable when
grown in Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum for 26 passages according to a 3T6 protocol (not shown).
The mature erythrocytes in the cardiac chambers of
Rce1
/
embryos did not suggest a severe
defect in hematopoiesis (Fig. 4, A and C).
However, because deficiencies in Ras proteins have been associated with
reduced hematopoiesis (35), we further assessed hematopoiesis in the
setting of Rce1 deficiency by transplanting Rce1
/
fetal liver hematopoietic stem cells
into lethally irradiated mice. There were no gross differences in the
appearance of fetal livers of Rce1
/
,
Rce1+/
, and Rce1+/+
embryos at days 14.5-15.5 post coitus. However, the number of nucleated cells isolated from Rce1
/
livers
was reduced by 37% (range, 12-68% in seven experiments) compared
with those from Rce1+/
and
Rce1+/+ embryos. Although the absolute number of
nucleated cells was less in Rce1
/
embryos,
there were no obvious functional deficits in those cells. The
dose-response relationship for colony-forming unit
granulocyte-macrophage progenitor colony formation from fetal liver
cells was similar over a range of granulocyte-macrophage
colony-stimulating factor concentrations in all three Rce1
genotypes. Cultures of Rce1
/
fetal liver
cells stimulated with saturating amounts of growth factor yielded
normal-to-increased numbers of colony-forming unit granulocyte-macrophage colonies, a subset of which showed a
distinctive morphology characterized tight clumping of the cells.
Rce1
/
fetal liver cells fully reconstituted
hematopoiesis in all 20 irradiated recipients. Peripheral blood cell
counts (hemoglobin, platelets, lymphocytes, neutrophils, and monocytes)
were normal at 1 month after adoptive transfer and were identical in
recipients that were injected with
Rce1
/
, Rce1+/
, or
Rce1+/+ cells (not shown). Blood cell counts
remained normal for more than 4 months after adoptive transfer, and
Southern blotting of genomic DNA from peripheral blood leukocytes
confirmed that Rce1
/
fetal liver cells
durably restored hematopoiesis in irradiated recipients (not shown).
Rce1 Deficiency Shifts the Electrophoretic Mobility of Ras
Proteins--
To determine whether the mouse Rce1 gene
participates in the post-translational processing of Ras proteins, we
compared the electrophoretic mobility of the Ras proteins from
Rce1
/
, Rce1+/+, and
Rce1+/
embryo lysates on SDS-polyacrylamide
gels. If the Rce1 gene product were involved in the
endoproteolytic processing of Ras, we reasoned that the electrophoretic
mobility of the unprocessed Ras proteins in
Rce1
/
embryo lysates would likely be
retarded. Indeed, this proved to be the case (Fig.
5, A and B).
Furthermore, Ras proteins with a normal electrophoretic mobility were
undetectable in the Rce1
/
lysates, even
after prolonged exposures of the blots. These results strongly suggest
that the Rce1 gene product is essential for Ras endoproteolytic processing (at least in E14.5 mouse embryos). Interestingly, the absence of proteolytic processing appeared to have
no effect on the levels of Ras proteins in the cells. Also, the
electrophoretic mobility of the Ras proteins was identical in
Rce1+/+ and Rce1+/
embryo lysates, suggesting that heterozygosity for the Rce1
knockout mutation does not affect Ras processing. To confirm that Ras
proteins from Rce1+/+ and
Rce1
/
cells truly had different
electrophoretic mobilities, we mixed equal amounts of lysates from
Rce1
/
and Rce1+/+
embryos and then performed a Western blot for Ras. As expected, two
distinct Ras bands were observed in the mixture (Fig. 5C). We also analyzed the electrophoretic mobility of Ras proteins that had
been immunoprecipitated from E14.5 embryos and cell lysates with a
pan-Ras antibody. Once again, homozygosity for the Rce1 knockout allele resulted in a retarded electrophoretic mobility of the Ras proteins (Fig. 5D).

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Fig. 5.
Abnormal electrophoretic mobility of Ras
proteins in lysates from Rce1 / embryos
and primary embryonic fibroblasts. A, a Western blot of
lysates of Rce1+/+,
Rce1+/ , and Rce1 /
embryos. Lysates were size-fractionated by SDS-polyacrylamide gel
electrophoresis, and a Western blot was performed with a
pan-Ras-specific monoclonal antibody, Ab-4. B, a Western
blot of lysates of Rce1+/+,
Rce1+/ , and Rce1 /
embryos with a Ki-Ras-specific monoclonal antibody (Ab-1).
C, a Western blot of an equal mixture of
Rce1+/+ and Rce1 /
embryo lysates with antibody Ab-4, demonstrating the expected doublet
band. D, abnormal electrophoretic mobility of Ras proteins
immunoprecipitated from lysates of Rce1 /
embryos and fibroblasts. Ras proteins from
Rce1+/+ and Rce1 /
embryos and cultured fibroblasts were immunoprecipitated with a pan-Ras
specific antibody; the immunoprecipitate was then analyzed on a Western
blot of an SDS-polyacrylamide gel with a pan-Ras-specific
antibody.
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|
Absence of CAAX Endoprotease Activity in Rce1
/
Fibroblasts--
The abnormal electrophoretic mobility of the Ras
proteins strongly suggested that the Rce1 gene product
was responsible for Ras endoproteolytic processing. If so,
carboxymethylation of Ras proteins from
Rce1
/
cells would be defective because
methylation requires prior removal of the three C-terminal amino acid
residues. To test that prediction, we metabolically labeled
Rce1+/+ and Rce1
/
fibroblasts with
L-[methyl-3H]methionine and
[35S]cysteine, immunoprecipitated the Ras proteins, and
assessed the methylation status of the Ras proteins after fractionating the immunoprecipitates on an SDS-polyacrylamide gel. The amount of
[3H]methanol released from the gel slice containing
20-23-kDa proteins by base hydrolysis (which reflects the extent of
carboxymethylation of the C-terminal prenyl cysteine) was substantially
greater in the Rce1+/+ immunoprecipitates than
in the Rce1
/
immunoprecipitates (1013 versus 239 dpm); analysis of 35S incorporation
levels showed comparable amounts of Ras protein in the
immunoprecipitates from Rce1
/
cells and
Rce1+/+ cells (2380 arbitrary units
versus 2150 arbitrary units, respectively). In two
subsequent experiments, we also observed more base-releasable [3H]methanol from Ras proteins from
Rce1+/+ cells than in those from
Rce1
/
cells (1557 versus 492 dpm
in one experiment, and 349 versus 45 dpm in the other).
These data suggested a defect in the carboxymethylation (and thus
proteolysis) of Ras proteins in the Rce1
/
fibroblasts.
To directly assess the endoproteolysis of Ras proteins by
Rce1+/+ and Rce1
/
fibroblasts, we used an in vitro assay with prenylated
recombinant proteins. Initially, we examined the ability of membrane
and cytosolic fractions from Rce1+/+ and
Rce1
/
fibroblasts to proteolytically process
farnesylated Ki-Ras. No proteolysis of the prenylated protein by the
cytosolic fraction of Rce1+/+ cells was
observed, but the membrane fraction contained robust proteolytic
processing activity (Fig. 6A).
In contrast, no detectable processing of the farnesylated protein was
observed with either the membrane or cytosolic fractions from
Rce1
/
fibroblasts. In membranes from
Rce1+/+ fibroblasts, prenyl protein endoprotease
activity increased linearly with increasing membrane protein
concentration, whereas in membranes from
Rce1
/
fibroblasts, activity was negligible
at all concentrations tested (Fig. 6B). Farnesyl-Ha-Ras,
farnesyl-N-Ras, the farnesyl-G
1 subunit of transducin,
geranylgeranyl-Ki-Ras, and geranylgeranyl-Rap1B were each found to be
substrates for the protease activity in the membranes from
Rce1+/+ fibroblasts, but processing of all these
prenylated proteins by the membranes of
Rce1
/
fibroblasts was either absent or near
the detection limit for this assay (Fig. 6C). The apparent
differences in the extent of processing of the different prenylated
proteins should not be interpreted as indicating any major differences
in their abilities to be recognized by the protease, because, except
for Ki-Ras, none of the proteins was purified after in vitro
prenylation, and hence their concentrations in the assay varied.

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Fig. 6.
Direct assays of the ability of
Rce1 / fibroblasts to
endoproteolytically process prenylated CAAX
proteins. A, an assay of the ability of cytosolic
and membrane fractions from Rce1 /
fibroblasts to endoproteolytically process farnesyl-Ki-Ras. Membrane
and cytosolic fractions (80 µg) from Rce1+/+
and Rce1 / fibroblasts were incubated for 30 min at 37 °C with farnesyl-Ki-Ras (2 µM) in 100 mM Hepes, pH 7.5, and 5 mM MgCl2.
The processed farnesyl-Ki-Ras was then methylated with 5.0 µM [3H]S-adenosylmethionine (1.5 Ci/mmol) and 20 µg of membranes from Sf9 insect
cells expressing high concentrations of the yeast prenyl protein
carboxymethyltransferase STE14. [3H]Methyl-prenylated
proteins were collected on filters, and methylation was quantified by
scintillation counting. Proteolysis is described as the number of pmol
[3H]methyl groups transferred to the prenylated protein.
Each data point was corrected by subtracting levels of
[3H]methyl groups trapped on filters in the absence of
the recombinant prenylated protein. These experiments were performed
twice, with each data point representing duplicate assays.
B, concentration dependence of prenyl protein protease
activity in the membrane fractions from Rce1+/+
and Rce1 / fibroblasts. In these experiments,
0-80 µg of fibroblast membranes was analyzed with 2.0 µM farnesyl-Ki-Ras as the prenyl protein substrate.
C, endoprotease activity in membranes from
Rce1+/+ and Rce1 /
fibroblasts against a panel of prenylated proteins. The prenyl group
attached to proteins is indicated by "f-" for farnesyl
or by "gg-" for geranylgeranyl. The -AAX
sequence for each protein is indicated in italics. The
concentrations of farnesyl-Ki-Ras and geranylgeranyl-Ki-Ras (determined
by a protein assay) were 1.0 µM. The approximate concentration (determined by exhaustive proteolysis
and methylation of each substrate) of geranylgeranyl-Rap1B was 0.2 µM, and the approximate concentrations of
farnesyl-Ha-Ras, farnesyl-N-Ras, and farnesyl-G 1 were 1 µM.
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Rce1 Deficiency Results in a Mislocalization of Ras
Proteins--
To test whether the absence of Ras proteolytic
processing and methylation altered the intracellular localization of
Ras, we compared the relative levels of Ras proteins in the membrane
and cytosolic fractions of Rce1
/
and
Rce1+/+ embryonic fibroblasts with
immunoblotting techniques. In spontaneously immortalized fibroblasts,
virtually all of the Ras in Rce1+/+ cells was
located in the membrane fractions (Fig.
7). In contrast, approximately 50% of
the Ras within Rce1
/
cells was in the
cytosolic fraction (Fig. 7). This experiment was repeated with early
passage primary embryonic fibroblasts. Once again, there was an
increased percentage of the Ras proteins in the cytosolic fractions of
Rce1
/
cells (not shown).

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Fig. 7.
Intracellular localization of Ras proteins in
immortalized Rce1+/+ and
Rce1 / fibroblasts. Cells were
fractionated into cytosolic (S100) and membrane
(P100) fractions by ultracentrifugation. Ras proteins were
immunoprecipitated from the cytosolic and membrane fractions, as well
as from the total cellular lysates, and then analyzed on a Western blot
of an SDS-polyacrylamide gel with the pan-Ras-specific antibody
Ab-4.
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To further address the issue of Ras localization within cells, both
Rce1
/
and Rce1+/+
cells were transfected with a green fluorescent protein-Ras fusion protein in which the C-terminal 18 amino acids were identical to mouse
Ki-Ras (including the CAAX sequence). The fluorescence was
highly localized to the plasma membrane in
Rce1+/+ cells (Fig.
8, A-C) but was largely
cytoplasmic in the Rce1
/
cells (Fig. 8,
D-F). As a control, we transfected EGFP into both Rce1+/+ and Rce1
/
cells. As expected, the green fluorescent protein was distributed uniformly in the cytoplasm of both cell types (Fig. 8,
G-I).

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Fig. 8.
Confocal microscopic images of
Rce1+/+ fibroblasts
(A-C) and Rce1 /
fibroblasts (D-F) transfected with a plasmid encoding an
EGFP-Ki-Ras fusion protein. G,
Rce1+/+ fibroblasts transfected with the
parental EGFP plasmid; H and I,
Rce1 / cytosolic fibroblasts transfected with
the EGFP plasmid.
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DISCUSSION |
Our studies show that the Rce1 gene product is
essential for the endoproteolytic processing of all of the mammalian
Ras proteins. Three lines of evidence support this statement. First,
Ras proteins with a normal electrophoretic mobility (indicating normal
processing) were undetectable in Rce1
/
embryos or fibroblast lysates. Second, metabolic labeling experiments revealed that the Ras proteins from Rce1
/
fibroblasts could not be carboxymethylated, a processing step that
requires prior proteolysis. Third and most important, the endoproteolytic processing of prenylated recombinant Ras proteins by
membranes from Rce1-
/
fibroblasts was undetectable.
Two findings from the in vitro assays of endoproteolytic
processing deserve comment. First, the membranes from
Rce1-deficient fibroblasts were defective in processing both
farnesylated and geranylgeranylated proteins, indicating that this
protease recognizes both classes of prenylated CAAX
proteins. Second, neither geranylgeranylated Rap1B nor farnesylated
G
1 was processed by Rce1
/
fibroblast membranes, strongly suggesting that the Rce1 gene product plays a broad role in processing prenylated CAAX proteins.
The principal finding our study, that Rce1 is critical for the
processing of a group of prenylated proteins, is entirely consistent with the conclusions of a companion study by Otto and co-workers (33).
They cloned the human RCE1 cDNA and expressed it at high levels in Sf9 insect cells by infection with a
recombinant baculovirus. The membrane preparations derived from the
transfected Sf9 cells exhibited a high level of
prenyl protease activity and were capable of processing both
farnesylated and geranylgeranylated proteins, including the Ras
proteins, Rap1B, and the heterotrimeric G protein G
1 subunit.
Whether the product of the Rce1 gene processes all of the
mammalian CAAX proteins is not known. In yeast, Afc1p
cleaves the -AAX from the precursor of the
a-mating factor. Recently, Tam and co-workers (20) reported
the cDNA sequence of the human homologue of yeast AFC1
and demonstrated that the human Afc1 protein has the capacity to
process yeast a-mating factor and to complement the mating
defect of yeast lacking both Afc1p and Rce1p. However, a human
homologue for yeast a-mating factor has not been identified,
and no substrates for the human AFC1 gene product are known. Defining
the unique and/or overlapping biochemical specificities for the
mammalian Afc1 and Rce1 proteins in processing mammalian
CAAX proteins will be of great interest.
A deficiency in Rce1 gene expression in mice is incompatible with
survival. The vast majority of Rce1
/
embryos
die late in gestation, and the few mice that are born alive are
severely runted and die shortly after birth. It is not clear why the
Rce1
/
embryos do not survive, in view of the
fact that all of the major organ systems developed and were free of
obvious pathology. Mutations that cause death late in mouse embryonic
development often involve disorders of the circulation (e.g.
defective hematopoiesis or a defective heart or placenta) (36).
However, the placenta of Rce1-deficient embryos appeared
normal; the liver contained large numbers of hematopoietic precursor
cells; the heart and great vessels were filled with mature
erythrocytes; the heart was normal in size; and the cardiac myocytes
appeared histologically normal. In addition, the rescue of lethally
irradiated mice by Rce1
/
hematopoietic stem
cells makes it unlikely that the Rce1-deficient mice die
from hematopoietic failure. Although the precise cause of death of
these animals remains unclear, we suggest that the failure to process
some population of prenylated CAAX proteins might render
certain cellular metabolic functions inefficient, perhaps in a
tissue-specific manner, leading to death without pathological changes
that are obvious by light microscopy.
In yeast, a deficiency in Rce1p interferes with Ras2p processing,
causes a mislocalization of a Ras2p-green fluorescent protein fusion to
the cytoplasm, and blocks the heat sensitivity caused by a mutationally
activated Ras protein. However, the deficiency in Rce1p does not affect
the growth of yeast under normal conditions (19). An absence of Rce1p
in yeast likely modulates Ras function rather than blocking it
completely. Of note, the rce1
yeast grew normally,
whereas the deletion of both yeast Ras genes (RAS1 and RAS2) is lethal (37). Also, the mislocalization of the Ras2p fusion in rce1
yeast did not appear to be absolute; a
fraction of the Ras2p fusion reached the plasma membrane (19). Our
studies of Rce1-deficient mice suggest intriguing
similarities to the situation in rce1
yeast. As in
rce1
yeast, the processing of Ras proteins in
Rce1-deficient embryos and fibroblasts was completely blocked, and Ras proteins were mislocalized within cells. Also, the
elimination of Rce1 enzymatic activity had no obvious effects on
cellular growth. Indeed, the growth of cultured
Rce1
/
fibroblasts was quite normal, and the
growth of Rce1
/
mouse embryos appeared to be
normal through E15. The relatively normal development of the
Rce1
/
mouse embryos indicated that Ras
function was probably not completely blocked, because a knockout of the
Ki-Ras gene alone causes death between E13 and E15 (35, 38), and a
deficiency of both Ki-Ras and N-Ras caused defective hematopoiesis and
embryonic death around E12 (35). Thus, as in yeast, we suggest that
Rce1 deficiency in mice modulates rather than blocks Ras
function. The precise extent to which Rce1 deficiency
interferes with Ras function in mammalian cells is not clear and
requires further study; however, the experimental tools developed in
this study, including the Rce1-deficient fibroblasts and the
adoptive bone marrow transfer model, should be helpful in addressing
that issue. For example, competitive repopulation assays will allow us
to ascertain if inactivation of Rce1 confers a proliferative
defect in specific lineages of hematopoietic cells.
Mutations that lead to constitutively active Ras proteins cause
increased cell growth and division and are involved in the pathogenesis
of many human cancers (39). The frequent involvement of mutationally
activated Ras proteins in human cancers, combined with the requirement
for C-terminal post-translational processing for Ras function, has
prompted efforts to interfere with the processing of the Ras proteins
and thereby impede the growth of cancers. Most of this effort has
focused on developing drugs to block protein farnesyltransferase
(FTase) and thereby interfere with the farnesylation of Ras. Potent
inhibitors of FTase have been developed and have proved to be
effective, both in cell culture systems and in animal models, in
retarding the growth of a wide variety of cancers. However, some Ras
proteins, including Ki-Ras, the one most commonly activated by somatic
mutation in human cancers, are geranylgeranylated by protein
geranylgeranyltransferase I in the setting of FTase inhibitors (40).
This alternate pathway for prenylation might limit the efficacy of
FTase inhibitors against many cancers. The development of Rce1
inhibitor drugs could provide another approach for reducing the
activity of Ras and would be expected to interfere with the processing
of the Ras proteins regardless of whether they were farnesylated or
geranylgeranylated. Of course, it remains to be determined whether any
particular Rce1 inhibitor drug would be clinically effective, but the
normal blood counts in mice rescued with Rce1-deficient
hematopoietic cells suggest that Rce1 inhibitor drugs might be free of
one side effect of many anticancer drugs, myelosuppression.
Interestingly, FTase inhibitors have been effective against cancers
that lack a mutationally activated Ras, suggesting that inhibiting the
activity of other farnesylated proteins, aside from Ras proteins, may
be important in inhibiting tumor growth (41). Those observations are
intriguing and suggest that inhibitors of Rce1 might be
active in blocking tumor growth (either alone or in combination with
FTase inhibitors) by acting against a range of different
CAAX proteins.