Amino Acid Residues That Define Both the Isoprenoid and
CAAX Preferences of the Saccharomyces
cerevisiae Protein Farnesyltransferase
CREATING THE PERFECT FARNESYLTRANSFERASE*
Brian Erich
Caplin
§,
Yoshikazu
Ohya¶, and
Mark S.
Marshall
§
**
From the
Walther Oncology Institute, Indianapolis,
the Departments of § Biochemistry and Molecular Biology and
Medicine, Division of Hematology and Oncology, Indiana
University School of Medicine, Indianapolis, Indiana 46202-5121, and the ¶ Department of Biology, Faculty of Science, University of
Tokyo, Hongo, Tokyo 113, Japan
 |
ABSTRACT |
Studies of the yeast protein farnesyltransferase
(FTase) have shown that the enzyme preferentially farnesylates proteins
ending in CAAX (C = cysteine, A = aliphatic
residue, X = cysteine, serine, methionine, alanine) and to
a lesser degree CAAL. Furthermore, like the type I protein
geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate
methionine- and leucine-ending substrates both in vitro and
in vivo. Substrate overlap of FTase and GGTase I has not
been determined to be biologically significant. In this study, specific
residues that influence the substrate preferences of FTase have been
identified using site-directed mutagenesis. Three of the mutations
altered the substrate preferences of the wild type enzyme
significantly. The ram1p-74D FTase farnesylated only
Ras-CIIS and not Ras-CII(M,L), and it geranylgeranylated all three
substrates as well or better than wild type. The
ram1p-206DDLF FTase farnesylated Ras-CII(S,M,L) at wild
type levels but could no longer geranylgeranylate the Ras-CII(M,L)
substrates. The ram1p-351FSKN FTase farnesylated Ras-CIIS
and Ras-CIIM but not Ras-CIIL. The ram1p-351FSKN FTase was
not capable of geranylgeranylating the Ras-CII(M,L) substrates, giving
this mutant the attributes of the dogmatic FTase that only farnesylates
non-leucine-ending CAAX substrates and does not
geranylgeranylate any substrate. These results suggest that the
isoprenoid and protein substrate specificities of FTase are
interrelated. The availability of a mutant FTase that lacked substrate
overlap with the protein GGTase-I made possible an analysis of the role
of substrate overlap in normal cellular processes of yeast, such as
mating and growth at elevated temperatures. Our findings suggest that
neither farnesylation of leucine-ending CAAX substrates nor
geranylgeranylation by the FTase is necessary for these cellular
processes.
 |
INTRODUCTION |
One recently discovered protein post-translational modification in
eukaryotic organisms is protein isoprenylation (1, 2). Protein
isoprenylation is the first in a series of post-translational modifications which serve to increase the hydrophobicity of the modified proteins, producing membrane association for proteins that
otherwise lack membrane binding structures (3, 4). Prenylated proteins
are modified by either a 15-carbon isoprenoid, farnesyl, or a 20-carbon
isoprenoid, geranylgeranyl. The carboxyl-terminal amino acid sequence
of the modified proteins serves to direct the addition of either of
these isoprenoids.
Three general classes of COOH-terminal sequences serve as substrates
for the protein prenyltransferases. The first class comprises those
that conform to the consensus sequence -CAAX, where C is cysteine, a is an aliphatic amino acid, and X is Ala, Cys,
Glu, Met, Ser, or Val (5). Proteins ending in -CAAX
sequences which are modified with farnesyl include the mammalian H-Ras
(CVLS), K-Ras (CVIM), and lamin B (CAIM) well as the yeast a-factor
(CVIA), Ras1p (CIIC), Ras2p (CIIS), and Rho4p (CIIM) proteins. The
second class is that of proteins that terminate in the consensus
sequence CAAL, which includes such proteins as the yeast
Rho2p (CIIL), Rsr1p (CTIL), and Rho1p (CVLL) proteins. The third class
of COOH-terminal motifs includes the sequence -CC or -CXC
found in the Rab family GTPases, which are double geranylgeranylated.
Two additional modifications follow the isoprenylation of
-CAAX and -CAAL proteins. The COOH-terminal tripeptide is endoproteolyzed, and the exposed isoprenyl-cysteine is
carboxylmethylated. Isoprenylation and the subsequent
carboxylmethylation serve to increase the hydrophobicity of the
modified protein, allowing it to associate physically with
intracellular membranes (4). Protein prenylation is catalyzed by at
least three distinct protein prenyltransferases: protein
farnesyltransferase (FTase),1
the type I protein geranylgeranyltransferase (GGTase-I), and the type
II protein geranylgeranyltransferase (GGTase-II) (5-8, 37). GGTase-II
transfers geranylgeranyl pyrophosphate to protein substrates that
have carboxyl-terminal sequences of -CXC or -CC. FTase and
GGTase-I transfer either farnesyl diphosphate (Fpp) or
geranylgeranyl diphosphate (GGpp) to protein substrates with carboxyl-terminal -CAAX and -CAAL
sequences, respectively. FTase and GGTase-I have been
identified in organisms as diverse as yeast (9-12), rat (6, 8, 13, 14,
37), and man (15). FTase and GGTase-I are heterodimeric enzymes
containing a common
subunit and highly related
subunits (11).
The conservation of enzyme structure and substrate preferences of these
enzymes suggests that they share a common catalytic mechanism. Kinetic
studies indicate that both FTase and GGTase-I preferentially bind
isoprenoid before binding the -CAAX or -CAAL
protein substrate (16, 17). Furthermore, both enzymes catalyze product
formation from ternary complex (isoprenoid substrate, protein
-CAAX or -CAAL substrate, and enzyme) faster than
the release of isoprenoid substrate (17, 18), suggesting the
conservation of catalytic mechanism of the two enzymes.
On the basis of their similar subunit composition, the tertiary
structures of FTase and GGTase-I are likely to be quite high. In
addition to their identical
subunits, the
subunits of FTase and
GGTase-I share approximately 35% amino acid sequence identity. Perhaps
not surprisingly, these enzymes have measurable isoprenoid and protein
substrate overlap in vitro and in vivo (19-21).
Substrate overlap between FTase and GGTase-I is particularly evident
with substrate proteins that terminate in CIIM; these substrates are geranylgeranylated by both FTase and GGTase-I (20). FTase can farnesylate protein substrates ending in the sequence CIIL, and GGTase-I farnesylates the yeast RhoB, which has a carboxyl-terminal CKVL sequence (21). Yeast FTase and GGTase-I enzymes also show substrate overlap in vivo, where Ras proteins in yeast that
lacks FTase are farnesylated by GGTase-I, and yeast that lacks
GGTase-I has a faster growth rate when FTase is overexpressed (19).
Mutational analysis of both the
and
subunits of FTase indicates
that each subunit plays a role in enzymatic catalysis (22-24). The
subunit is important for stabilization of the heterodimer and may also
directly affect the catalytic reaction (25). Mutagenesis of a
Zn2+-coordinating Cys residue in the
subunit of FTase
indicates a role for this subunit in isoprenoid binding and a direct
role in enzymatic catalysis (26). Photoaffinity cross-linking of peptides to FTase indicates the presence of an intersubunit location for the catalytic active site (27, 28). Further studies of the FTase
subunit indicate that distinct NH2-terminal and
COOH-terminal regions of this subunit are involved in determining the
-CAAX specificity of this enzyme and that single site
mutations are sufficient to produce a mutant FTase that has an
increased affinity for -CAAL substrates (29).
The results from these and other studies suggest that the FTase
subunit determines both isoprenoid and protein substrate specificities.
This is supported logically by the fact that their
subunits are the
only difference between the FTase and GGTase-I enzymes. For this study
it was hypothesized that regions highly conserved among eukaryotic
FTase
subunits that differed from the known GGTase-I
subunits
contained the residues that determined a preference for farnesyl
pyrophosphate and CAA(A,S,C,M) over CAAL. Seven
such regions and individual residues were identified in the yeast FTase
subunit which met these criteria. Each site was changed from the
yeast FTase
subunit sequence to the corresponding yeast GGTase-I
subunit amino acid sequence. Mutations affecting residues 74, 206-212, and 351-354 in the
subunit altered -CAAX and
isoprenoid specificity upon reconstitution with the
subunit. The
ram1p-351FSKN FTase was surprising in that rather than
behaving more like the GGTase-I, it was found to behave as a dogmatic
FTase: able to farnesylate only non-leucine-ending -CAAX
proteins and unable to geranylgeranylate any substrate tested.
Expression and analysis of the ram1p-351FSKN mutant enzyme
in ram1 Saccharomyces cerevisiae yeast lacking the wild type
subunit of the FTase strongly suggested that the loose substrate
specificity observed for FTase in vitro and in vivo does not play a critical physiological function in S. cerevisiae.
 |
MATERIALS AND METHODS |
[3H]Fpp (15 Ci/mmol) and [3H]GGpp
(15 Ci/mmol) were purchased from American Radiolabeled Chemicals. All
other chemical reagents were from U. S. Biochemical Corp. and Sigma.
Restriction endonucleases were from New England Biolabs. GF/F filters
were direct from Whatman. BioSafe II scintillation mixture was from
Research Products International. Protein assay reagents for the
Bradford and BCA assays were purchased from Bio-Rad and Pierce,
respectively. HyperfilmMP was purchased from Amersham. DNA sequencing
was performed with Sequenase version 2.0 from U. S. Biochemical Corp.
YL1/2 antibody was purified from hybridoma cells (30). Construction of
Ras1-CAAX mutants and Ras protein expression and
purification are described elsewhere (5). Ras1-CAAX will be
referred to as Ras followed by the specific CAAX sequence,
for example Ras-CIIS. The S. cerevisiae strains used were
YO2A (MATa RAM1 ade2 lys2 leu2
trp1 his3 ura3), YO4D(MAT
RAM1 ade2 lys2 leu2 trp1
his3 ura3), YO3C (MATa ram1:LEU2 ade2 lys2 leu2 trp1 his3
ura3), and YO5C (MATa ram1:LEU2 ade2
lys2 leu2 trp1 his3
ura3), PT1 (MAT
).
Site-directed Mutagenesis of the RAM1 Gene--
Specific amino
acid changes in the RAM1 protein were made in pMM101 (pRAM1-EEF) as
described (30). The mutations were confirmed by DNA sequencing. The
oligonucleotides used are as follows, named according to the
amino acid changes produced in the RAM1 protein: R57HV60FL61FS63RV64H
(5'-ATCATCGTATATCTCTAAGTGACGTTGGAAAAATTTGTAATGTGCTTCTGTAGTGTCGGT-3'); I74D (5'-GAGAGCAGGTTCATCATTCTTTTCATCATC-3');
G206DV208DT210L G212F
(5'-GCTCAAGGCACAGTATATAAATCTTAGATGTCTTCATCGACTTCTAAACAGGTCTT-3'); G308T (5'-AAAACTATAGCAAGTGTCAACAAGTTTGTT-3');
L351FR352
SD353KK354N
(5'-TGAGTGGGCCCCTGGATTCTTACTAAAACCAGGTTGCTCTTTTTC-3');
C367G (5'-CAGTCCTAATAGGCCATAATTTGTATGGTA-3');
S385K (5'-CTTAATATTATGTGGTTTATCATTAGGAGT-3').
Mutations will be referred to by the amino acid changes produced
in the Ram1 protein, for example the
R57HV60FL61FS63RV64H
changes will be referred to as Ram1p-57HFFRH. All RAM1
mutagenic oligonucleotides were synthesized by an Oligo 1000 DNA
Synthesizer from Beckman Instruments Inc.
Construction of RAM1 Yeast Expression Vectors--
The yeast
shuttle vector YCP50-ADH containing a 5'-EcoRI cloning site
and a 3'-XbaI cloning site was designed to express the RAM1, RAM1-351FSKN, and
RAM1-74D genes with a constitutive alcohol
dehydrogenase promoter. Plasmids YCP50-ADH-RAM1,
YCP50-ADH-RAM1-351FSKN, and
YCP50-ADH-RAM1-74D were produced by subcloning
EcoRI/XbaI fragments of the appropriate vectors
according to the method of Maniatis et al. (38). The identity of the vectors was confirmed by restriction endonuclease digestion with EcoRI and XbaI enzymes and dideoxy
sequencing (U. S. Biochemical Corp., Sequenase version 2.0).
Expression of Prenyltransferases in Escherichia
coli--
Coexpression of each protein prenyltransferase was
accomplished by the cotransformation of E. coli strain
DH5
with 0.1 µg each of pBH57 (pRAM2) and pMLM4 (pCDC43),
pMM101-3 (pRAM1), pBC27-3 (pRAM1-57HFFRH), pBC22-81
(pRAM1-74D), pBC26-13 (pRAM1-206DDLF),
pBC25-7 (pRAM1-308T), pBC30-5
(pRAM1-351FSKN), pBC21-41 (pRAM1-367G), or
pBC24-8 (pRAM1-385K)(32) and selection on Luria Broth
plates containing 100 mg/ml ampicillin and 10 mg/ml chloramphenicol.
Recombinant FTase and GGTase-I enzyme expression in E. coli
and cell lysate preparation was performed as described previously (20,
31, 33).
Isoprenylation of Ras-CAAX Substrates with Crude Bacterial
Lysates and Purified Enzymes--
Prenyltransferase assays were
carried out and analyzed as described previously (20, 31, 33). Kinetic
studies were performed with purified wild type and
ram1p-351FSKN FTases using 0.5-20 µM
Ras-CII(S,M) or 1.0-38 µM Ras-CIIL for protein
substrates and 0.021-20 mM [3H]-Fpp or
[3H]-GGpp. In a standard FTase assay, the reaction was
terminated after 10 min by the addition of 0.9 ml of 1 M
HCl in ethanol. After protein precipitation for 15 min at room
temperature, samples were slow filtered over pre-wet Whatman GF/F
filters and washed four times with 2-ml volumes of 100% ethanol.
Filters were air dried and scintillation counted in 5 ml of BioSafe II
liquid scintillation mixture.
Time course assays for both the wild type and ram1p-351FSKN
FTases with each substrate combination were performed to determine the appropriate incubation times necessary to acquire initial rate
values. Kinetic data were collected from at least four separate experiments for each substrate. Steady-state kinetic parameters were
determined by fit to an ordered Bi-Bi steady-state reaction mechanism
using GraFit version 3.02 (Erithacus Software, Inc., 1996). Equation 1
is for such a model (34).
|
(Eq. 1)
|
Purification of Wild Type FTase and ram1p-351FSKN
FTase--
DH5
cells cotransformed with the RAM2 and either
RAM1 or RAM1-351FSKN genes were used
to inoculate 1-liter volumes of Luria Broth with 100 mg/ml ampicillin
and 10 mg/ml chloramphenicol. Protein expression and cell pellets were
obtained according to the protocol outlined above. Cell pellets were
resuspended into 10 ml of purification lysis buffer (20 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 4 mM
dithiothreitol, 20 µM ZnCl2, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 mM
leupeptin, 0.1 µM pepstatin A) and lysed by sonication
using five 30-s pulses with a Fisher model 300 sonicator at 35% power.
Lysates were cleared by centrifugation at 14,000 × g
at 4 °C. Cleared lysates were loaded onto three sequential 5-ml
Econo Q columns (Bio-Rad) at a flow rate of 0.5 ml/min. The columns
were washed with TDZ buffer (20 mM Tris-HCl, pH 7.5, 4 mM dithiothreitol, and 20 µM
ZnCl2). The column was eluted with a 10% step gradient
from 0 to 100% TDZN buffer (TDZ buffer plus 1 M NaCl).
Prenyltransferase activity eluted in the 200-500 mM NaCl
fractions. The active fractions were pooled and concentrated in Amicon
Centriprep 10 concentrators. Wild type FTase and
ram1p-351FSKN FTase enzyme purification was confirmed by
SDS-polyacrylamide gel electrophoresis, Coomassie Brilliant Blue
(U. S. Biochemical Corp.) staining, and Bio-Rad Bradford protein
analysis.
The wild type FTase and the ram1p-351FSKN FTase enzymes
prepared by Econo Q purification were prepared for Glu-Glu-Phe
immunoaffinity purification adapted from Refs. 12 and 35. Concentrated
active fractions from Econo Q columns were diluted with TDZE (TDZ + 0.1 mM EDTA) to bring the final salt concentration to 100 mM NaCl. Protease inhibitors were added (0.2 mM
phenylmethylsulfonyl fluoride, 0.1 mM leupeptin, 0.1 µM pepstatin A) (YL1/2 loading buffer). The active
fraction was loaded onto a Gamma Bind Plus/YL1/2 antibody column at a
flow rate of 0.05 ml/min in a continuous loop overnight at 4 °C. The
preparation of this column has been described elsewhere (12). The
column was washed with 10 volumes of TDZE at a flow rate of 0.1 ml/min.
Column elution was achieved with YL1/2 elution buffer (TDZ + 5 mM Asp-Phe dipeptide; Sigma) at 0.1 ml/min; fractions were
0.5-ml volumes. The active fractions were assayed by the method of
Bradford (Bio-Rad) for protein content and by filter binding
prenyltransferase assays for activity. Active fractions were dialyzed
at 4 °C, in 250 ml of 50 mM Na-HEPES, pH 7.5, 20 mM MgCl2, 20 µM
ZnCl2, 2 mM dithiothreitol, and 15% glycerol. The dialysate was separated into 0.05-ml aliquots and flash frozen in
N2 (liquid) and stored at
80 °C until ready for use.
The presence of prenyltransferase was confirmed by 10%
SDS-polyacrylamide gel electrophoresis and a combination of Coomassie
Brilliant Blue staining and Western blot analysis with YL1/2
(anti-tubulin (EEF)) primary antibody and Amersham horseradish
peroxidase (anti-rat) secondary antibody. The presence of
immunoreactive bands was determined by ECL detection (Amersham).
Purified enzymes were used for Michaelis-Menten kinetic analysis as
described above.
Yeast Cell Growth Curves and Mating Assays--
Yeast cells
YO2A, YO4D, YO3C, and YO5C were transformed by the lithium acetate
method (CLONTECH). YO3C and YO5C cells are each
transformed with pMJ13 (YCP50-ADH), pBC31
(YCP50-ADH-RAM1-351FSKN), pBC32
(YCP50-ADH-RAM1), or pBC33
(YCP50-ADH-RAM1-74D). Transformants were
selected on SD-Ura plates at 30 °C to produce strains BC3CY
(YCP50-ADH, MATa), BC3CY31
(YCP50-ADH-RAM1-351FSKN, MATa), BC3CY32
(YCP50-ADH-RAM1, MATa), BC3CY33
(YCP50-ADH-RAM1-74D, MATa), BC5CY (YCP50-ADH,
MAT
), BC5CY31 (YCP50-ADH-RAM1-351FSKN, MAT
), BC5CY32
(YCP50-ADH-RAM1, MAT
), BC5CY33
(YCP50-ADH-RAM1-74D, MAT
). Transformants
were grown in 5 ml of SD-Ura selective medium overnight at 30 °C,
200 rpm. The cell number was determined by counting with a
hemacytometer. These overnight cultures were used to inoculate cultures
for growth curves. 1 × 106 cells of each transformant
were seeded in 25 ml of SD-Ura broth. Cultures were grown at either 28 or 35 °C, 300 rpm, and 0.8-ml samples were collected. Cell growth
was determined by measuring A600 at indicated
time points. The cell number was determined by hemacytometry and was
found to be linear with respect to optical density.
Mating assays consisted of BC3CY, BC3CY31, BC3CY32, and BC3CY33 cells
(MATa) each mated to PT1 cells (MAT
). Briefly, an equal number of
MATa cells, which had been sonicated to separate individual cells, was
mixed with a 10-fold excess of MAT
cells and filtered onto
individual nitrocellulose filters. MATa and MAT
cells on filters
were incubated on YPD plates for 4 h at 30 °C. After incubation cells were diluted in minimal media and sonicated. Mated cells were
plated onto minimal agar plates. Revertant controls were subjected to
the same conditions as the mated cells, and the cell number control
experiments were plated onto minimal agar plates with nutrients adenine
(20 mg/ml), lysine (30 mg/ml), tryptophan (20 mg/ml), and histidine (20 mg/ml). A detailed description of yeast mating assays can be found in
Ref. 39.
 |
RESULTS |
Identification of Amino Acid Residues Conserved among Eukaryotic
FTase
Subunits but Not Conserved with the
Subunits of the
GGTase-I--
The
subunits of FTase and GGTase-I sequences were
aligned using the CLUSTAL version 1.6 multiple sequence alignment
program (Baylor College of Medicine, Houston). A visual search strategy was employed to identify amino acid residues potentially important for
determining the unique substrate preferences of the two enzymes. Briefly, amino acid residues conserved between both enzymes were presumed to be necessary for the tertiary structure of the enzymes. Amino acid residues specifically conserved in only the FTase or the
GGTase-I
subunits, but not conserved for both enzymes, were hypothesized to determine the distinct substrate specificities of the
two enzymes. Seven such regions or individual residues were identified
and selected for mutagenesis. The amino acid residues of the yeast
FTase Ram1p
subunit were substituted with the corresponding residues of the yeast Cdc43p GGTase-I
subunit. The following RAM1
mutants were prepared: RAM1-57HFFRH, RAM1-74D,
RAM1-206DDLF, RAM1-308T,
RAM1-351FSKN, RAM1-367G, and
RAM1-385K. The result of this search strategy and the
substitutions made in the FTase enzyme are depicted in Fig.
1. The conserved sequences that met the
criteria of the search strategy generally were found to be embedded
within the context of short stretches of conservation for both the
FTase and GGTase-I enzymes.

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Fig. 1.
Sequence alignment of protein
isoprenyltransferases with amino acid substitutions. subunits
of yeast and mammalian FTases and GGTase-I were aligned using CLUSTAL
version 1.6. Regions of sequence conservation in the FTase subunit
were replaced by the corresponding residues of the GGTase subunit.
Amino acid substitutions to the yeast Ram1p are indicated in
bold.
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|
Substrate Preferences of Mutant FTase Enzymes--
Recombinant
wild type FTase was expressed in E. coli, and the bacterial
lysate was assayed for its ability to farnesylate or geranylgeranylate
the Ras-CIIS, Ras-CIIM, and Ras-CIIL proteins (Fig.
2). The wild type FTase enzyme
efficiently farnesylated Ras-CIIS, Ras-CIIM, and Ras-CIIL and also
geranylgeranylated Ras-CIIM (31). The quantity of
[3H]farnesyl and [3H]geranylgeranyl
incorporated into each Ras substrate by the wild type FTase under
standardized conditions was determined by time course assays to
represent the initial rate of reaction and was considered the 100%
control value for comparison with the mutant FTase enzymes. Extracts
were prepared from E. coli expressing each mutant ram1p
FTase: ram1p-57HFFRH, ram1p-74D,
ram1p-206DDLF, ram1p-308T,
ram1p-351FSKN, ram1p-367G, and
ram1p-385K. Each was assayed for the ability to
isoprenylate Ras-CIIS, Ras-CIIM, and Ras-CIIL with
[3H]farnesyl pyrophosphate (Fig.
3A) and
[3H]geranylgeranyl pyrophosphate (Fig.
3B).

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Fig. 2.
Substrate specificity of recombinant wild
type FTase. Recombinant wild type FTase was expressed in E. coli, and 50 µg of bacterial lysate was assayed in the presence
of either 67 pmol of [3H]Fpp or 67 pmol of [3H]GGpp and 5 µM Ras-CIIS (black bars), Ras-CIIM
(shaded bars), or Ras-CIIL (stripped bars)
proteins.
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Fig. 3.
Relative substrate preferences of mutant subunit FTases. Recombinant mutant FTase enzymes were expressed in
E. coli, and 50 g of bacterial lysate was assayed in
the presence of 67 pmol of [3H]Fpp (panel A) or 67 pmol of
[3H]GGpp (panel B) and 5 µM Ras-CIIS
(black bars), Ras-CIIM (shaded bars), or Ras-CIIL
(stripped bars) proteins. Data are expressed as percent of
wild type FTase activity.
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The effect of the mutations could be divided into three categories: 1)
wild type activity and substrate specificity, 2) impaired activity, and
3) altered substrate specificity. In the first category were the
ram1p-367G and ram1p-385K mutants that showed
nearly wild type farnesylation and geranylgeranylation activity for
each of the substrates tested. The second class of mutants included the
ram1p-57HFFRH and ram1p-308T mutants that
exhibited a general impairment of isoprenyl transferase activity. The
third class of mutations included ram1p-74D,
ram1p-206DDLF, and ram1p-351FSKN, each of which
showed a unique set of substrate specificities. The
ram1p-74D mutant was restricted to farnesylating only the
Ras-CIIS protein while retaining the ability to geranylgeranylate each
Ras-CAAX protein tested. The ram1p-74D mutant
demonstrated a nearly 3-fold increase in ability to geranylgeranylate Ras-CIIM. The ram1p-206DDLF mutant enzyme was no longer
able to use geranylgeranyl as a substrate, yet it still farnesylated
each of the Ras-CAAX proteins including the Ras-CIIL
protein. The third mutant enzyme of this class,
ram1p-351FSKN, was unique in that it was still able to
farnesylate Ras-CIIS and Ras-CIIM but not Ras-CIIL. Furthermore, the
ram1p-351FSKN enzyme had no capacity to geranylgeranylate
any of the Ras-CAAX proteins. By the criteria established
before these studies, the ram1p-351FSKN FTase mutant was an
ideal FTase in that it was unable to farnesylate Ras-CIIL and had no
geranylgeranyltransferase activity.
To confirm that these unusual results were not artifacts based upon
assaying the enzymes in bacterial lysates, the wild type, and
ram1p-308T, ram1p-351FSKN mutants were
expressed in E. coli and purified by a combination of ion
exchange and immunoaffinity chromatography. SDS-PAGE analysis of
purified enzymes indicates that these proteins purify equally well by
this approach. Purified enzymes displayed substrate specificities and
activity comparable to those observed in the cell lysates (data not
shown).
Steady-state Kinetic Analysis of Wild Type Ram1p and
ram1p-351FSKN FTase--
The altered substrate specificity
of the mutant ram1p-351FSKN FTase suggested that there were
fundamental changes in substrate recognition by the mutant enzyme. To
determine the nature of these changes, steady-state kinetic analysis
was performed using purified wild type Ram1p and
ram1p-351FSKN FTases. Both enzymes were analyzed using a
combination of Ras-CIIS, Ras-CIIM, Ras-CIIL, and either Fpp or GGpp as
substrate. The data were fit to several bisubstrate models using GraFit
version 3.02 (Robin Leatherbarrow, Erithacus Software, 1996) and were found to fit best (F test and reduced
2) to an ordered
Bi-Bi kinetic model and as such are consistent with the findings with
the mammalian FTase (16, 18).
Table I depicts the kinetic relationships
between the affinity for isoprenoid (Fpp or GGpp) and the affinity for
the Ras-CAAX substrates. A clear preference for Fpp is
observed for both the wild type enzyme and the
ram1p-351FSKN enzyme, where
Km(farnesyl) is <1 µM,
and the Km(geranylgeranyl) is >3.5
µM. The ram1p-351FSKN mutant enzyme has at
least a 4-fold reduction in affinity for GGpp over the wild type
enzyme. The Km and Kd of GGpp for
the ram1p-351FSKN mutant enzyme are 17.5 and 43.5 µM, and 3.59 and 7.61 µM for the wild type
Ram1p FTase. Additionally, the ram1p-351FSKN mutant enzyme
is reduced in its affinity for Ras-CIIL, with a
Km(CIIL) of 22.8 µM
versus 10.2 µM for wild type FTase. Analysis
of the Kcat data indicate that although the
Kcat values of the wild type Ram1p and
ram1p-351FSKN enzymes are similar for the combination of
farnesyl and either Ras-CIIS or Ras-CIIM substrates, the
ram1p-351FSKN FTase is clearly impaired relative to the
wild type enzyme when utilizing either GGpp and Ras-CIIM or Fpp and
Ras-CIIL. These results are consistent with those seen in the standard
isoprenyltransferase assays (Fig. 3, A and
B).
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Table I
Kinetic constants for the wild type Ram1p and mutant
Ram1p-351FSKN FTase enzymes
Kinetic constants were determined by best fit to an ordered Bi-Bi
steady-state reaction (Equation 1). Substrate concentrations for
[3H]farnesyl diphosphate and [3H]geranylgeranyl
diphosphate were 0.021-20 µM, and Ras-CAAX
concentrations were 0.5-38 µM. Kinetic constants were
determined by GraFit version 3.02 (Erithacus Software, Inc., 1996).
Abbreviations: W.T., wild type; Km(iso),
Km for isoprenoid;
Km(CaaX) Km
for Ras-CAAX substrate; Kd(iso),
Kd for isoprenoid; Kcat,
intrinsic catalytic rate at saturating substrate concentrations; ND,
not able to be determined.
|
|
RAM1-351FSKN Restores Yeast Mating and Growth at
Elevated Temperatures--
The overlapping substrate preferences of
the yeast FTase and GGTase-I suggest the possibility that the FTase may
farnesylate CAAL-ending proteins or geranylgeranylate other
CAAX substrates in vivo. With a restricted
ability to only farnesylate non-leucine-ending CAAX
substrates, the ram1p-351FSKN FTase provided a tool to
address this question genetically. Using this mutant, we determined the
ability of a "perfect" FTase to complement the growth and mating
defect of ram1-1 S. cerevisiae cells. S. cerevisiae strains BC3CY[ram1],
BC3CY31[ram1p-351FSKN], BC3CY32 [Ram1p], and
BC3CY33[ram1p-74D] were grown at either a permissive
(28 °C) or restrictive (35 °C) temperature in minimal medium
(Fig. 4, A and B,
respectively). Each strain grown at the permissive temperature showed
similar growth kinetics, with doubling times of approximately 6 h
in mid-log phase, reaching stationary phase approximately 28 h
postinoculation. Strains grown at the restrictive temperature showed
dramatically different growth characteristics. The BC3CY32[Ram1] and
BC3CY31[ram1p-351FSKN] both grew at the nonpermissive
temperature with similar doubling times in mid-log phase of
approximately 5 h demonstrating full functional complementation of
the growth defect by the "perfect" FTase. The YO3C[ram1-1] yeast
strain grew only poorly at the restrictive temperature (36), with a
doubling times of 12 h but never showing logarithmic growth. The
substrate specificity mutant, ram1p-74D FTase, was tested
for complementation of the growth defect of BC3CY[ram1-1]. We
observed that this mutant was incapable of restoring growth at the
restrictive temperature and in fact grew more poorly than the parental
ram1:LEU2 strain (Fig. 4B).

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Fig. 4.
Complementation of the temperature-sensitive
growth defect of in ram1-1 S. cerevisiae. Yeast was
transformed with constitutive expression vectors YCP50-ADH (open
squares), YCP50-ADH-RAM1 (open circles),
YCP50-ADH-RAM1-351FSKN (open triangles), or
YCP50-ADH-RAM1-74D (asterisks) and
grown at either 28 °C (panel A) or 35 °C (panel
B). The number of cells was found to be linear at
A600 nm, and cell growth was determined by
optical density measurements.
|
|
The same mutant
subunit FTase enzymes were evaluated for
restoration of mating in the ram1:LEU2 strain (Table
II). Multiple steps in mating
responsiveness require isoprenylated proteins. Mating assays of
S. cerevisiae (ram1-1 MATa) expressing
RAM1, RAM1-74D, or
RAM1-351FSKN
subunit indicate that both
mutant FTases are capable of prenylating the protein substrates
necessary for efficient mating.
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|
Table II
Mating assays to test for complementation of the mating defect in
ram1-1 yeast
MATa S. cerevisiae with the
ram1-1::LEU2 allele were transformed with
constitutively expressed vectors YCP50-ADH, YCP50-ADH-RAM1,
YCP50-ADH-RAM1-351FSKN, or
YCP50-ADH-RAM1-74D. Each was mated with PT1 MAT
yeast. The percent of mated cells versus the total number of
viable MATa cells in the mating is depicted in the table. The data are
representative of four separate matings.
|
|
 |
DISCUSSION |
The alignment of primary amino acid sequences of enzymes with
similar catalytic properties is a time-tested approach to identifying those amino acid residues that distinguish one enzyme from another. Using a simple strategy for identifying amino acid residues that may be
important for the substrate specificity differences observed between
the FTase and GGTase-I enzymes, we constructed seven Ram1p
subunit mutants containing one to five residue substitutions each. The
recombinant expression mutant ram1p and wild type Ram2p proteins to
form FTase heterodimers in E. coli proved to be a rapid
method for characterizing both isoprenoid and protein substrate preferences. In terms of relative activity and substrate preferences, the mutant FTases fell into three classes: wild type enzymes, catalytically dead enzymes, and altered substrate preference enzymes. The recent publication of the crystal structure of the rat FTase provides insight into the results observed for each of the these three
classes of mutations (25) (Fig. 5).

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Fig. 5.
C tracing of the wild type rat FTase with
ram1p-351FSKN mutations. The C trace of the rat
FTase (25) was redrawn. The location of the 351FSKN
mutation is depicted in bold. An asterisk
represents the position of the putative isoprenoid binding
pocket.
|
|
The first class of mutant enzymes displayed substrate preferences that
were similar to those of the wild type FTase enzyme. These include the
ram1p-367G and the ram1p-385K mutant enzymes.
In the
subunit, cysteine 367 is adjacent to tyrosine 366, believed
to be involved in forming the isoprenoid binding groove of the enzyme.
This substitution does not alter the positioning of this crucial
tyrosine residue. The conserved tyrosine residue at position 362 has
been suggested to be important for substrate binding (29). Despite the
close proximity of the ram1p-367G mutation to tyrosine 362, the FTase had the same substrate specificity of the wild type FTase.
The location of the ram1p-385K substitution in the
three-dimensional structure of the FTase appears to have substrate
preferences and catalytic properties similar to those of the wild type
enzyme, as only the farnesylation of the Ras-CIIL substrate is
impaired. This is likely caused by the positioning of this residue on a
tail structure that is located on a face of the enzyme which is
opposite that of the substrate binding pocket (25). The rat FTase x-ray
structure reveals this region of the enzyme to be involved in
intersubunit contacts, and it may be important for maintaining proper
positioning of the
and
subunits. Clearly, this region has only
a modicum of influence on the substrate preference of the enzyme.
Those mutant enzymes that were affected catalytically can be explained
structurally using the rat FTase crystal structure as a model. The
ram1p-57HFFRH substitution is located in the first
helix of the enzyme. The affected portion of this
helix is located
on the surface of the protein adjacent to the Ras-CAAX
binding groove. Although these same residues are found in the wild type
GGTase-I enzyme they may not provide the proper amino acid context to
form the first
helix of the
subunit, which may be required for
proper protein substrate binding (25). The ram1p-308T
mutant is more easily understood in terms of the possible cause of its
catalytic impairment. Residue 308 of the wild type Ram1p is a glycine
residue that is located at the beginning of an
helix that contains
two hydrophobic residues proposed to be critical for the positioning of
the isoprenoid substrate. Additionally, cysteine 309 adjacent to
glycine 308 corresponds to cysteine 299 of the rat FTase which
coordinates the catalytic Zn2+ ion (26). Substitution of a
glycine residue with a threonine residue can reduce the conformational
flexibility of a polypeptide chain due to the additional stearic
hindrance of the threonine side chain. The
helix secondary
structure that positions cysteine 309 for Zn2+ coordination
and isoprenoid binding is likely to be disrupted by the limited
conformations of the polypeptide chain which are imposed by the
presence of the threonine side chain.
The third class of
subunit mutants that were produced using this
strategy possessed altered isoprenoid and protein substrate specificities. The ram1p-74D substitution is located in a
short loop between helix 1 and helix 2 of the rat FTase, and it is
positioned away from the putative active site of the enzyme (25). The
wild type residue is isoleucine. It is unlikely that this residue
interacts directly with the CAAX box of the protein
substrate. The substitution of a charged residue for a branched chain
hydrophobic amino acid possibly effects the local structure of the
enzyme sufficiently to perturb the substrate binding pocket.
Mechanistically, the coupled changes in both protein and isoprenoid
substrate preferences of this mutant suggest that the particular amino
acid at the X position of the CAAX sequence and
isoprenoid selected to modify that protein substrate are tightly interrelated. Although the mutant enzyme modifies Ras-CIIS with farnesyl nearly as well as the wild type Ram1p FTase, it cannot farnesylate either Ras-CIIM or Ras-CIIL. The geranylgeranylation activity of this mutant is not hindered for either protein substrate and in the case of Ras-CIIM is significantly increased. The placement of this mutation outside the binding pocket of the enzyme and its
unique substrate preferences suggest that regions outside the
isoprenoid binding pocket can influence the substrate preferences for
the FTase enzyme.
The enhanced ability of the ram1p-206DDLF mutant to
distinguish between Fpp and GGpp suggests that these residues are
important for distinguishing between the different sized isoprenoids.
Examination of the rat FTase crystal structure places this substitution
in a position that lines the putative isoprenoid binding pocket (25). Arginine 211 of the
subunit is positioned opposite the
Zn2+ ion and may be important for coordination of the
pyrophosphate group of the isoprenoid. The ram1p-206DDLF
substitution is located in the highly conserved region and may serve to
lock the position of the conserved arginine so that only Fpp is
functionally positioned into the isoprenoid binding pocket.
The ram1p-351FSKN mutant FTase provided the most
interesting insight into the substrate recognition by the FTase. This
mutant enzyme showed that both isoprenoid substrates and protein
substrates can be distinguished accurately with only a few changes to
the amino acid sequence of the wild type enzyme. This enzyme behaves as
a "perfect" FTase, it farnesylates only non-leucine-ending substrates, and it cannot geranylgeranylate any of the substrates tested. Steady-state kinetic analysis was employed to investigate the
unique substrate preferences of the ram1p-351FSKN at a
kinetic level.
Table I depicts the enzymatic rate constants for both the wild type and
the ram1p-351FSKN mutant FTases. The
ram1p-351FSKN FTase was shown to have a reduced affinity
(increased Km) for the GGpp and Ras-CIIM substrate
combination (17.5 and 1.87 µM, respectively)
versus the wild type FTase (3.59 and 0.28 µM, respectively). Additionally, the ram1p-351FSKN has a
reduced affinity for Ras-CIIL relative to the wild type enzyme (22.8 versus 10.2 µM, respectively). Interestingly, with Ras-CIIL as a protein substrate the most dramatic effect is
observed with the Kcat values, the catalytic
turnover rates at saturating concentrations of substrates. The wild
type enzyme can turnover product, farnesylated-Ras-CIIL, nearly six
times faster than the ram1p-351FSKN mutant FTase (0.35 versus 0.06, respectively). Together these data support the
findings from Fig. 3, A and B, where the
ram1p-351FSKN mutant FTase cannot geranylgeranylate the
Ras-CIIM substrate, nor can it farnesylate the Ras-CIIL substrate under
the standard assay conditions.
Based on the kinetic data in Table I, a model of substrate preference
can be proposed. In an ordered Bi-Bi reaction system, the first
substrate, the isoprenoid, must bind to the active site of the enzyme.
Comparison of the Km for isoprenoid for the wild
type enzyme with Fpp indicates that the affinity of the enzyme for this
substrate is only slightly dependent on the Ras-CAAX substrate that is used in the reaction. Clearly, GGpp is a less favored
substrate. To form a productive binary complex the isoprenoid must
remain bound to the enzyme. Table I indicates that Fpp is released from
the enzyme more readily when Ras-CIIS is the protein substrate than
when either Ras-CIIM or Ras-CIIL is the protein substrate. Isoprenoid
binding and release constitute the first step of the catalysis
reaction, the binding of the isoprenoid to form an active
enzyme-isoprenoid binary complex. The second step in the ordered Bi-Bi
reaction would be the binding of the CAAX protein substrate.
Table I indicates that a protein preference order is Ras-CIIS
Ras-CIIM > Ras-CIIL with Fpp as the isoprenoid substrate. The
next step in substrate preference and isoprenyl transfer is at the
level of catalytic rate, where the rates are Fpp-Ras-CIIS > GGpp-Ras-CIIM
Fpp-Ras-CIIM > Fpp-Ras-CIIL. Clearly, these
rates themselves do not represent the transfer activities found in Fig.
2. The substrate preference of the wild type Ram1p and the
ram1p-351FSKN mutant enzymes is apparently the result of a
complex interrelation among the affinity and release for the isoprenoid
substrate, the affinity for the CAAX substrate, and the
catalytic rate for the transfer reaction.
The limited substrate specificity of the ram1p-351FSKN
FTase mutant provided a genetic tool with which to address the question of the physiological role of the substrate overlap between the FTase
and the GGTase-I. S. cerevisiae requires a functional FTase for growth at elevated temperatures. The discovery of a "perfect" FTase (ram1p-351FSKN) that can only farnesylate
non-leucine-ending protein substrates allowed us to determine which
property of the FTase is essential for viability. The
ram1p-74D FTase mutant, which has only limited
farnesylation activity and normal geranylgeranylation activity,
provided a readily available control for these experiments. The poor
farnesylation activity of the ram1p-74D FTase mutant
suggests that it is unlikely to farnesylate those substrates that are
required for growth at elevated temperatures. The mating assays
indicate that only farnesylation of a limited set of -CAAX
substrates is necessary for normal mating. Although these two mutants
have been tested with only a limited set of Ras-CAAX
substrates, these studies indicate that farnesylation, and not
geranylgeranylation, is required for growth of yeast at elevated
temperatures and a functional mating pathway. We observe from these
studies that not only are there residues that specify isoprenoid
preference (ram1p-206DDLF) and protein preference
(ram1p-74D), but there are also regions of the
subunit
in which both isoprenoid and protein substrate preferences are
determined (ram1p-351FSKN). These studies have shown that
the recognition of isoprenoid substrate and protein substrate is
interrelated, suggesting that the specific isoprenoid bound to the
enzyme influences which CAAX substrate will be bound. The
availability of these mutant enzymes with specific isoprenoid and
protein preferences also provides specific genetic tools for
determination of the role that farnesylation or geranylgeranylation of
particular proteins may play in normal cellular processes.
 |
FOOTNOTES |
*
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: 1044 W. Walnut St., Rm.
351, Indianapolis, IN 46202. Tel.: 317-274-7565; Fax: 317-274-7592;
E-mail: mark_marshall{at}iucc.iupui.edu.
1
The abbreviations used are: FTase,
farnesyltransferase; GGTase, geranylgeranyltransferase; Fpp, farnesyl
phosphate; GGpp, geranylgeranyl diphosphate; ADH, alcohol
dehydrogenase.
 |
REFERENCES |
-
Glomset, J. A.,
Gelb, M. H.,
and Farnsworth, C. C.
(1990)
Trends Biochem. Sci.
15,
139-142[CrossRef][Medline]
[Order article via Infotrieve]
-
Gibbs, J. B.
(1991)
Cell
65,
1-4[Medline]
[Order article via Infotrieve]
-
Ghomashchi, F.,
Zhang, X.,
Liu, L.,
and Gelb, M. H.
(1995)
Biochemistry
34,
11910-11918[Medline]
[Order article via Infotrieve]
-
Parish, C. A.,
and Rando, R. R.
(1996)
Biochemistry
35,
8473-8477[CrossRef][Medline]
[Order article via Infotrieve]
-
Moores, S. H.,
Schaber, M. D.,
Mosser, S. D.,
Rands, E.,
O'Hara, M. B.,
Garsky, V. M.,
Marshall, M. S.,
Pompliano, D. L.,
and Gibbs, J. B.
(1991)
J. Biol. Chem.
266,
14603-14610[Abstract/Free Full Text]
-
Reiss, Y.,
Goldstein, J. L.,
Seabra, M. C.,
Casey, P. J.,
and Brown, M. S.
(1991)
Cell
62,
81-88
-
Manne, V.,
Roberts, D.,
Tobin, A.,
O'Rourke, E.,
De Virgilio, M.,
Meyer, C.,
Ahmed, N.,
Kurtz, B.,
Resh, M.,
Kung, H. F.,
and Barbacid, M.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
7541-7545[Abstract]
-
Seabra, M. C.,
Reiss, Y.,
Casey, P. J.,
Brown, M. S.,
and Goldstein, J. L.
(1991)
Cell
65,
429-434[Medline]
[Order article via Infotrieve]
-
He, B.,
Chen, P.,
Chen, S. Y.,
Vancura, K. L.,
Michaelis, S.,
and Powers, S.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
4448-4452[Abstract]
-
Finegold, A. A,
Johnson, D. I.,
Farnsworth, C. C.,
Gelb, M. H.,
Judd, S. R.,
Glomset, J. A.,
and Tamanoi, F.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
4448-4452[Abstract]
-
Ohya, Y.,
Goebl, M.,
Goodman, L. E.,
Petersen-Bjorn, S.,
Friesen, J. D.,
Tamanoi, F.,
and Anraku, Y.
(1991)
J. Biol. Chem.
266,
12356-12360[Abstract/Free Full Text]
-
Mayer, M. P.,
Prestwich, G. D.,
Dolence, J. M.,
Milano, P. D.,
Wu, H.,
and Poulter, C. D.
(1993)
Gene (Amst.)
132,
41-47[CrossRef][Medline]
[Order article via Infotrieve]
-
Chen, W.,
Andres, D. A.,
Golstein, J. L.,
Russell, D. W.,
and Brown, M. S.
(1991)
Cell
66,
327-334[Medline]
[Order article via Infotrieve]
-
Yokoyama, K.,
and Gelb, M. H.
(1993)
J. Biol. Chem.
268,
4055-4060[Abstract/Free Full Text]
-
Zhang, F. L.,
Diehl, R. E.,
Kohl, N. E.,
Gibbs, J. B.,
Giros, B.,
Casey, P. J.,
and Omer, C. A.
(1994)
J. Biol. Chem.
269,
3175-3180[Abstract/Free Full Text]
-
Pompliano, D. L.,
Rands, E.,
Schaber, M. D.,
Mosser, S. D.,
Neville, J. A.,
and Gibbs, J. B.
(1992)
Biochemistry
31,
3800-3807[Medline]
[Order article via Infotrieve]
-
Yokoyama, K.,
McGeady, P.,
and Gelb, M. H.
(1995)
Biochemistry
34,
1344-1354[Medline]
[Order article via Infotrieve]
-
Furfine, E. S.,
Leban, J. J.,
Landavazo, A.,
Moomaw, J. F.,
and Casey, P. J.
(1995)
Biochemistry
34,
6857-6862[Medline]
[Order article via Infotrieve]
-
Trueblood, C. E.,
Ohya, Y.,
and Rine, J.
(1993)
Mol. Cell. Biol.
13,
4260-4275[Abstract]
-
Caplin, B. E.,
Hettich, L. A.,
and Marshall, M. S.
(1994)
Biochim. Biophys. Acta
1205,
39-48[Medline]
[Order article via Infotrieve]
-
Armstrong, S. A.,
Hannah, V. C.,
Goldstein, J. L.,
and Brown, M. S.
(1995)
J. Biol. Chem.
270,
7864-7868[Abstract/Free Full Text]
-
Andres, D. A.,
Goldstein, J. L.,
Ho, Y. K.,
and Brown, M. S.
(1993)
J. Biol. Chem.
268,
1383-1390[Abstract/Free Full Text]
-
Ohya, Y.,
Caplin, B. E.,
Qadota, H.,
Tibbetts, M. F.,
Anraku, Y.,
Pringle, J. R.,
and Marshall, M. S.
(1996)
Mol. Gen. Genet.
252,
1-10[CrossRef][Medline]
[Order article via Infotrieve]
-
Mitsuzawa, H.,
Esson, K.,
and Tamanoi, F.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
1704-1708[Abstract]
-
Park, H.-W.,
Boduluri, S. R.,
Moomaw, J. F.,
Casey, P. J.,
and Beese, L. S.
(1997)
Science
257,
1800-1804
-
Fu, H.,
Moomaw, J. F.,
Moomaw, C. R.,
and Casey, P. J.
(1996)
J. Biol. Chem.
271,
28541-28548[Abstract/Free Full Text]
-
Ying, W.,
Sepp-Lorenzino, L.,
Cai, K.,
Aloise, P.,
and Coleman, P. S.
(1994)
J. Biol. Chem.
269,
470-477[Abstract/Free Full Text]
-
Pellicena, P.,
Scholten, J. D.,
Zimmerman, K.,
Creswell, M.,
Huang, C. C.,
and Miller, W. T.
(1996)
Biochemistry
35,
13494-13500[CrossRef][Medline]
[Order article via Infotrieve]
-
Villar, K. D.,
Mitsuzawa, H.,
Yang, W.,
Sattler, I.,
and Tamanoi, F.
(1997)
J. Biol. Chem.
272,
680-687[Abstract/Free Full Text]
-
Kilmartin, J. V.,
Wright, B.,
and Milstein, C.
(1982)
J. Cell Biol.
93,
576-582[Abstract]
-
Caplin, B. E.,
and Marshall, M. S.
(1995)
Methods Enzymol.
250,
51-68[Medline]
[Order article via Infotrieve]
-
Hanahan, F.
(1985)
in
DNA Cloning: A Practical Approach (Glover, D. M., ed), Vol. 1, p. 109, IRL Press, Oxford
-
Mayer, M. L.,
Caplin, B. E.,
and Marshall, M. S.
(1992)
J. Biol. Chem.
267,
20589-20593[Abstract/Free Full Text]
-
Segel, I. H.
(1993)
Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, pp. 560-565, John Wiley and Sons, New York
-
Skinner, R. H.,
Bradley, S.,
Brown, A. L.,
Johnson, N. J. E.,
Rhodes, S.,
Stammers, D. K.,
and Lowe, P. N.
(1991)
J. Biol. Chem.
266,
14163-14166[Abstract/Free Full Text]
-
Caplan, A. J.,
Tsai, J.,
Casey, P. J.,
and Douglas, M. G.
(1992)
J. Biol. Chem.
267,
18890-18895[Abstract/Free Full Text]
-
Reiss, Y.,
Seabra, M. C.,
Armstrong, S. A.,
Slaughter, C. A.,
Goldstein, J. L.,
and Brown, M. S.
(1991)
J. Biol. Chem.
266,
10672-10677[Abstract/Free Full Text]
-
Maniatis, T.,
Fritsch, E. F.,
and Sambrook, J.
(1982)
Molecular Cloning: A Laboratory Manual, pp. I.53-I.73, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Marshall, M.,
Mahoney, D.,
Rose, A.,
Hicks, J. B.,
and Broach, J. R.
(1987)
Mol. Cell. Biol.
7,
4441-4452[Medline]
[Order article via Infotrieve]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.