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(Received for publication, May 17, 1995; and in revised form, June 26, 1995) From the
Protein prenylation utilizes different types of isoprenoids
groups, namely farnesyl and geranylgeranyl, to modify proteins. These
lipophilic moieties attach to carboxyl-terminal cysteine residues to
promote the association of soluble proteins to membranes. Most
prenylated proteins are geranylgeranylated. Geranylgeranylation is
catalyzed by two different prenyltransferases, the type I and type II
geranylgeranyl transferases, both of which utilize geranylgeranyl
diphosphate as a lipid donor. In the yeast Saccharomycescerevisiae, the BET2 gene encodes the
Protein prenylation is a post-translational lipid modification
that involves the covalent attachment of isoprenoid groups onto
cysteine residues at or near the carboxyl termini (Casey, 1992; Schafer
and Rine, 1992; Sinensky and Lutz, 1992). The attachment of a
lipophilic isoprenoid group to proteins is believed to increase their
hydrophobicity, allowing otherwise hydrophilic proteins to associate
with membranes. Up to 0.5% of total cellular proteins are estimated to
be prenylated (Epstein et al., 1991). Known prenylated
proteins include small GTP-binding proteins of the Ras superfamily,
nuclear lamins, the yeast mating pheromone a-factor, and trimeric G
proteins (Casey, 1992; Schafer and Rine, 1992; Sinensky and Lutz,
1992). These proteins are engaged in a variety of cellular processes,
which include the control of cell growth, signal transduction,
cytokinesis, and intracellular membrane traffic (Balch, 1990; Barbacid,
1987). Two different isoprenoid groups, farnesyl (15 carbons) and
geranylgeranyl (20 carbons), are post-translationally attached to
proteins (Epstein et al., 1991). Farnesyl is added to proteins
that terminate in a CAAX motif (where C is cysteine, A is an
aliphatic amino acid, and X can be methionine, cysteine,
alanine, glutamine, phenylalanine, or serine), while geranylgeranyl is
transferred onto proteins that end in CAAL (where L is leucine), CC, or
CXC motifs (X is any amino acid) (Reiss et
al., 1990, 1991; Seabra et al., 1991, 1992). Most known
prenylated proteins are geranylgeranylated (Epstein et al.,
1991). Farnesyl and geranylgeranyl groups are attached to proteins
from all-trans farnesyl diphosphate (FPP) ( FPP is the
product of the farnesyl diphosphate synthase. This enzyme, which is the
most abundant and widely occurring prenyltransferase, catalyzes the
formation of FPP by the sequential addition of isopentenyl diphosphate
(IPP) to dimethylallyl diphosphate (DMAPP), and geranyl diphosphate
(GPP) (Anderson et al., 1989; Bartlett et al., 1985;
Sheares et al., 1989). In some organisms, GGPP is synthesized
by a GGPP synthase that catalyzes stepwise additions of IPP to DMAPP,
GPP, and FPP. This type of GGPP synthase activity has been detected in
mammalian tissue. However, eukaryotic geranylgeranyl diphosphate
synthases are known that synthesize GGPP by the addition of a single
molecule of IPP to FPP (McCaskill and Croteau, 1993; Sagami et
al., 1992, 1993, 1994). But, due to its low activity and the
problems in separating this enzyme from FPP synthase, its purification
has proven to be difficult (Runquist et al., 1992; Sagami et al., 1985, 1993, 1994). GGPP is the substrate for two
different protein prenyltransferases, the type I (GGTase-I) and type II
(GGTase-II) geranylgeranyl transferases (Jiang and Ferro-Novick, 1994;
Seabra et al., 1991, 1992). GGTase-I catalyzes the transfer of
a geranylgeranyl group from GGPP onto proteins that terminate in a CAAL
motif, while GGTase-II attaches geranylgeranyl to terminal CC or
CXC residues. Its protein substrates include members of the
Rab family of small GTP-binding proteins (Jiang and Ferro-Novick, 1994;
Seabra et al., 1992). In the yeast Saccharomycescerevisiae, the GGTase-II is composed of three subunits,
which are encoded by BET2, BET4 (formerly called MAD2), and MRS6, respectively (Rossi et al.,
1991; Jiang and Ferro-Novick, 1994; Jiang et al., 1993; Li et al., 1993). The BET2 gene product binds to the
product of the BET4 gene to form the catalytic component of
this enzyme. MRS6 encodes the accessory protein that binds the
protein substrate. Mutations in these genes abolish the
geranylgeranylation of Ypt1p and Sec4p, two small GTP-binding proteins
that mediate intracellular membrane traffic (Jiang and Ferro-Novick,
1994; Li et al., 1993; Rossi et al., 1991). The bet2-1 gene is a recessive temperature-sensitive mutant
allele that fails to grow at 37 °C (Newman and Ferro-Novick, 1987).
In this mutant, a failure to geranylgeranylate Ypt1p and Sec4p leads to
a defect in the membrane association of these proteins. This deficiency
results in a block in intracellular membrane trafficking (Rossi et
al., 1991). In an attempt to identify new genes that may interact
genetically with BET2, BET4, or MRS6, we
isolated a suppressor of the bet2-1 mutant. This
suppressor gene, named BTS1, suppresses the growth defect of bet2-1 when expressed on a low (CEN) or high (2
µm) copy vector. Sequence analysis revealed a significant homology
between BTS1 and the geranylgeranyl diphosphate synthase from Neurosporacrassa, suggesting that BTS1 encodes the homologue of this gene in S.cerevisiae. In accordance with this proposal, the BTS1 gene product was found to be required for the membrane
attachment of Ypt1p and Sec4p, a process that is known to require
geranylgeranylation. When BTS1 was expressed in bacterial
cells, it generated an activity that was able to convert FPP to GGPP,
thereby conclusively demonstrating that the BTS1 gene product
is the yeast geranylgeranyl diphosphate synthase. This enzyme is a
previously unidentified component of the yeast isoprenoid biosynthetic
pathway.
The smallest group B plasmid (pS8) that we
isolated contained a 2.8-kb insert (Fig. 1b). To
analyze the ability of this insert to suppress bet2-1,
we cloned this fragment into a high copy URA3 vector (pRS426)
to generate pSJ28. When pSJ28 was transformed into bet2-1 mutant cells, suppression was significantly enhanced (Fig. 1, compare b and c to the mutant alone
in a). In fact, growth of the mutant was restored to that of
wild type (Fig. 1, compare c and d),
suggesting that suppression was gene dosage dependent.
Figure 1:
Suppression of the bet2-1 mutant is gene dosage dependent. bet2-1 cells
(ANY119) were transformed with either pS8 (CEN, URA3)
or pSJ28 (2 µm, URA3). The transformants were streaked
onto YPD plates and incubated at 37 °C for 3 days. a,
ANY119; b, ANY119 (pS8); c, ANY119 (pSJ28); d, SFNY26-6A (wild type).
Figure 2:
Overexpression of the suppressor gene
increases the membrane-bound pool of Ypt1p and Sec4p in bet2-1 mutant cells. Wild type (W.T.)
(SFNY26-6A) and bet2-1 mutant cells (ANY119) that
contained pSJ28 were grown to early exponential phase in minimal medium
that was supplemented with the appropriate amino acids and 2% gluocse.
Cells were harvested, converted to spheroplasts, lysed, and the
membrane (P) and soluble (S) fractions were recovered
by centrifuging the lysates (T) at 100,000
Figure 3:
Suppression analysis and sequence
strategy. A 1.6-kb SspI-NruI fragment that fully
suppresses the bet2-1 mutant was sequenced in both
directions using the strategy shown above. ORF, open reading
frame.
Figure 4:
The
nucleotide sequence and predicted amino acid sequence of BTS1.
The nucleotide sequence of the 1.6-kb SspI-NruI
fragment is shown above. The five conserved regions in all
known FPP and GGPP synthases are
indicated.
Figure 5:
A
comparison of the BTS1 and albino-3 (Al-3) gene
products. The amino acid sequence of Bts1p is compared to Al-3 using
the Bestfit program (GCG software package). Identity is indicated by a line, and conserved changes are marked by twodots (two corresponding bases in a codon) or onedot (one corresponding base in a codon) between the
sequences. The gaps are designated by dots within a sequence.
Bts1p and Al-3 share 40% identity at the amino acid
level.
Figure 6:
SFNY368 (
Figure 7:
The
membrane attachment of Ypt1p and Sec4p is defective in
Figure 8:
Reverse-phase HPLC elution profile of
radiolabeled prenyltransferase reaction mixture. The reaction mixtures
from the incubation of crude extracts of E. coli containing
pUC118 (opencircle) or pUC118/BTS1 (solidcircle) with
[1-
Figure 9:
Saturation curves for
[
Previously, we have shown that the yeast GGTase-II is
composed of three subunits (BET2, BET4, and MRS6). Bet2p, the The
function of BTS1 was revealed by analyzing the sequence of
this gene. The predicted amino acid sequence of Bts1p was found to be
significantly homologous to the albino-3 gene product, the N.crassa GGPP synthase (Carattoli et al.,
1991). Upon a closer examination, we also found that Bts1p contains
five highly conserved motifs that are present in all known FPP and GGPP
synthases (Chen and Poulter, 1994), including the aspartate-rich
sequences proposed to be involved in binding and catalysis (Ashby and
Edwards, 1990; Joly and Edwards, 1993; Song and Poulter, 1994). This
finding suggested that BTS1 encodes the yeast GGPP synthase.
To confirm this hypothesis, we expressed the BTS1 gene in
bacteria. Bacterial lysates that express Bts1p were found to contain an
activity that synthesizes GGPP from IPP and FPP. The suppression of
the bet2-1 mutant by BTS1 could be explained in
several ways. The BTS1 gene product may itself have GGTase-II
activity, or it could directly interact with GGTase-II to stimulate its
activity. In either situation, the overexpression of BTS1 would be expected to increase GGTase-II activity. However, this
was not observed. Alternatively, suppression may simply be a
consequence of increasing the intracellular pool of GGPP. Since in
vitro prenylation studies have demonstrated that mutant GGTase-II
has a low affinity (increased K Because each of the subunits of the GGTase-II are essential, we
anticipated that BTS1 would also be required for the
vegetative growth of yeast cells. To our surprise, the Since BTS1 is not
essential for the growth of yeast cells, the synthase gene may be
duplicated. Preliminary DNA hybridization experiments, however, argue
against this possibility. Another explanation for the dispensability of BTS1 is that GGTase-II might utilize FPP as an alternate
substrate. However, since GGTase-II cannot transfer FPP to Ypt1p, this
possibility seems unlikely (Jiang et al., 1993). Furthermore,
extracts prepared from
Volume 270,
Number 37,
Issue of September 15, pp. 21793-21799, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-subunit of the type II geranylgeranyl transferase. Mutations in
this gene cause a defect in the geranylgeranylation of small
GTP-binding proteins that mediate vesicular traffic. In an attempt to
analyze those genes whose products may interact with Bet2, we isolated
a suppressor of the bet2-1 mutant. This suppressor gene,
called BTS1, encodes the yeast geranylgeranyl diphosphate
synthase. BTS1 is not essential for the vegetative growth of
cells; however, disrupting it impedes the geranylgeranylation of many
cellular proteins and renders cells cold sensitive for growth. Our
findings imply that BTS1 suppresses the bet2-1 mutant by increasing the intracellular pool of geranylgeranyl
diphosphate.
)and all-trans
geranylgeranyl diphosphate (GGPP), respectively (Casey, 1992). These
lipid precursors are intermediates in the isoprenoid biosynthetic
pathway (Goldstein and Brown, 1990). This pathway consists of a series
of reactions by which mevalonate is converted into a diverse family of
lipophilic molecules that contain a repetitive five-carbon structure.
The isoprenoids are subsequently incorporated into a large number of
end products, which includes: sterols, ubiquinones, dolichols, tRNAs,
and prenylated proteins (Goldstein and Brown, 1990).
Strains, Media, and Growth
Conditions
The following strains were used in this study:
ANY119 (MAT
, bet2-1, ura3-52, his4-619), NY648 (MAT
a/, leu2-3, 112/leu2-3, 112, ura3-52/ura3-52), NY180 (MAT, ura3-52, leu2-3, 112), SFNY26-6A (MAT, his4-619), and SFNY368 (MAT, ura3-52, leu2-3, 112, URA3::BTS1). Yeast strains were grown at 25 or 37 °C in
either YP or selective minimal medium that was supplemented with 2%
glucose.Isolation of BTS1
The yeast genomic DNA
library used in this study was prepared by ligation of genomic DNA that
was prepared from ANY119. This DNA was partially digested with Sau3A and inserted into the BamHI site of pRS316 (CEN, URA3). The library was used to transform the bet2-1 mutant (ANY119), and the transformants were (1
10
) selected on minimal medium lacking uracil.
After a 3-day incubation at 25 °C, the cells were stamped onto YPD
plates and incubated overnight at 37 °C. 11 positive transformants
were obtained. Plasmids (pS1-pS11) retrieved from these transformants
were amplified in Escherichia coli and retested in ANY119.DNA Sequence and Analysis
The DNA
sequence of the BTS1 gene was determined by the
dideoxynucleotide chain termination method (Sanger et al.,
1977). The reactions were performed using the Sequenase (U. S.
Biochemical Corp.) protocol, and the data were analyzed with GCG
software. Homology searches were performed with the EMBO/GenBank and
Swiss-prot data bases.Disruption of the BTS1 Gene
To disrupt BTS1, a 1.7-kb DraI-NruI fragment containing
the BTS1 gene was excised from pS8 and cloned into the PvuII site of pUC118 to generate pSJ30. The plasmid-borne
disruption of BTS1 was constructed by replacing a 0.65-kb SacI-EcoRI fragment in pSJ30 with a 1.2-kb SacI-EcoRI fragment containing the URA3 gene. The resulting plasmid (pSJ31) was digested with SspI and BglII and transformed into NY648. The
transformants were sporulated, and tetrad analysis was performed. After
3 days at 25 °C, all 48 tetrads examined displayed two large and
two small colonies. The large colonies were Ura, and
the small ones were Ura
.
DNA Hybridization Analysis
Yeast genomic
DNA prepared from NY180 or SFNY368 was examined by DNA-DNA
hybridization as described before (Naumvoski and Friedberg, 1984).
Genomic DNA digested with BglII was fractionated on a 0.8%
agarose gel and transferred to a BioTrans membrane (ICN). The blot was
probed with a radiolabeled 0.65-kb SacI-EcoRI
fragment, containing BST1, prepared by random-primer labeling
and visualized by autoradiography.Cell Fractionation Studies
Wild type
(NY180) and the 
BTS1 strain (SFNY368) were grown
overnight at 30 °C in YPD medium to early exponential phase. 1
aliquot of cells (150 A units) was pelleted and
washed once with ice-cold 10 mM sodium azide. The remaining
cells were shifted to 14 °C, and the incubation was continued for
12 h before the cells were harvested. To generate spheroplasts, cells
were resuspended in 0.7 ml of 10 mM ice-cold sodium azide and
mixed with an equal volume of 2
spheroplast medium (2.8 M sorbitol, 100 mM Tris-HCl (pH 7.5), 20 mM sodium
azide) containing 100 units of zymolyase. After a 1-h incubation at 25
°C, the spheroplasts were harvested by centrifugation in a clinical
centrifuge during a spin at 1400 rpm for 5 min, washed, and lysed in
1.4 ml of ice-cold lysis buffer (0.8 M sorbitol, 10 mM triethanolamine (pH 7.2), 1 mM EDTA) as described before
(Rossi et al., 1991). Cell debris was removed during a 3-min
spin at 450 g, and the supernatant from this spin was
centrifuged at 100,000 g for 1 h to generate a soluble
fraction. The pellet was resuspended in a volume of lysis buffer equal
to the supernatant. Samples were electrophoresed and subjected to
Western blot analysis using anti-Ypt1p or anti-Sec4p antibodies (1:2000
dilution).Expression of the BTS1 Gene in E. coli
To
express BTS1 in E. coli, the BTS1 open
reading frame sequence was generated by polymerase chain reaction using
two primers that overlapped the initiation codon or the region 100 base
pairs downstream from the stop codon. EcoRI and ClaI
sites were also incorporated into the 5`- and 3`-ends, respectively.
The polymerase chain reaction product was digested with EcoRI
and ClaI and cloned into the pUC118 expression vector. The
resulting gene fusion encodes a Bts1 protein with six additional
NH
-terminal amino acids from -galactosidase. This
construct was then transformed into JM101 bacterial cells and expressed
as described before (Jiang and Ferro-Novick, 1994).Synthase Assay and Product Analysis
The
standard assay mixture contained 20 mM BHDA buffer (pH 7.0),
10 mM -mercaptoethanol, 1 mM MgCl,
0.1% (w/v) bovine serum albumin, 200 µM DMAPP or FPP, 20
µM [1-
C]IPP (10 µCi/µmol
purchased from Amersham), and 70-80 µg of protein in a total
volume of 200 µl. DMAPP, FPP, and GGPP were synthesized by the
method of Davisson et al.(1986). After 10 min at 37 °C,
200 µl of CH
OH-HCl (4:1) was added, and the incubation
was continued for 30 min. The reaction mixture was extracted with 1 ml
of ligroin, and 0.5 ml of the ligroin layer was mixed with 10 ml of
Cytoscint-ES (ICN) for the measurement of radioactivity in a Packard
Tri-Carb 4530 liquid scintillation spectrometer. Products were analyzed
using HPLC. For the product analysis, bovine serum albumin was omitted
from the standard assay mixture, but 10 mM sodium fluoride was
present to suppress phosphatase activity. After a 1-h incubation at 37
°C, the reaction was terminated by the addition of EDTA (12.5
mM, final concentration). Unlabeled GGPP (25 µg) was
added, and 150 µl of the mixture was injected onto a Shodex
Asahipak ODP-50 column (4.6 mm (inner diameter) 250 mm). 2-min
fractions were collected, and the radioactivity in each fraction was
determined by liquid scintillation counter after the addition of 15 ml
of Cytoscint-ES.Preparation of Yeast Extracts and Protein Prenylation
Assay
Yeast cells were grown in YPD medium at 25 °C to
late log phase. The cells were harvested, lysed with glass beads, and
centrifuged at 100,000 g for 45 min. The soluble
fraction was collected and assayed for GGTase-II activity. Prenylation
assays were performed in a 50-µl reaction that contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl, 5 mM dithiothreitol, 25 µg of extract, 0.4 µM of
recombinant Ypt1p, and varying concentrations of
GGPP (American Research Lab, 17,500 dpm/pmol).
The reaction mixture was incubated at 30 °C for 30 min before it
was terminated with 1 M HCl in ethanol (1 ml) and filtered on
a Whatman GF/A filter as described before (Jiang et al.,
1993).
Isolation of Suppressors of the bet2-1
Mutant
bet2-1 is a temperature-sensitive
mutant that grows at 25 °C (permissive temperature) but dies at 37
°C. To isolate genes whose products may interact with the Bet2
protein (Bet2p), we screened a yeast genomic library that was prepared
from the bet2-1 mutant for plasmids that conferred
growth at 37 °C. After screening 1 10 transformants, 11 positive colonies were obtained and retested.
The growth of mutant cells containing six of these plasmids (group A)
was indistinguishable from that of wild type at 37 °C (data not
shown). The other five (group B), however, did not suppress as well.
Restriction analysis indicated that the plasmids in group A contained
the BET2 structural gene. Since the genomic library was
prepared from bet2-1 mutant cells, the restoration of
growth observed at 37 °C is not true complementation. Plasmids in
group B contained an overlapping 2.0-kb region of DNA. Therefore, the
gene that suppresses the bet2-1 mutant is located within
this 2.0-kb fragment.
Plasmid pSJ28 Increases the Membrane-bound Pool of
Ypt1p and Sec4p in bet2-1 Mutant Cells
Previous
studies have shown that the membrane association of Ypt1p and Sec4p is
defective in bet2 mutant cells (Rossi et al., 1991).
This defect is a consequence of the failure to geranylgeranylate these
proteins (Jiang et al., 1993; Rossi et al., 1991).
Thus, the lethal phenotype of the bet2-1 mutant is
likely to be a consequence of the inability of these proteins to attach
to membranes. Since plasmid pSJ28 suppresses the growth defect of the bet2-1 mutant at 37 °C, it may also cure the
membrane attachment defect observed in these cells. To address this
possibility, we transformed pSJ28 into bet2-1. When the
distribution of Ypt1p and Sec4p was examined in these transformants and
compared to the mutant and wild type, pSJ28 was found to enhance the
membrane association of these small GTP-binding proteins (Fig. 2, compare the amount in the lysate (T) to the
supernatant (S) and pellet (P) fractions). The
presence of pSJ28 did not lead to an increase in the residual GGTase-II
activity that can be measured in bet2 mutant cells (Jiang et al., 1993). Thus, the restoration of the membrane
association of Ypt1p and Sec4p is not a consequence of increasing
GGTase-II activity.
g.
Samples were then subjected to Western blot analysis using anti-Ypt1p
and anti-Sec4p antibodies.
Cloning and Sequencing the Suppressor
Gene
To localize the suppressor within the 2.8-kb genomic
fragment described above, subclones of pSJ8 were constructed and
inserted into pRS316 (URA3, CEN). Suppression studies
revealed that the SacI site contained within this fragment is
critical for its activity. The smallest region of DNA capable of
suppressing bet2-1 was found to be a 1.6-kb SspI-NruII fragment. This region of DNA was sequenced
in both directions using the strategy shown in Fig. 3. An open
reading frame of 1005 base pairs that spans the SacI site was
identified. We called the gene that encodes this open reading frame BTS1 (Bet Two Suppressor). BTS1 is predicted to encode a protein of 335 amino acids with a
calculated molecular mass of 38,627 daltons (Fig. 4). Overall,
the amino acid composition of the Bts1p is hydrophilic, and no
significant hydrophobic stretches were observed.
Bts1p Is Homologous to Known
Prenyltransferases
Comparison of the predicted Bts1p amino
acid sequence with the Swiss-Prot protein sequence data base revealed a
significant similarity between Bts1p and the N.crassaalbino-3 gene product (Carattoli et al., 1991).
These proteins are 40% identical at the amino acid level with the most
conserved region localized to the middle of these proteins (Fig. 5). The albino-3 gene encodes a geranylgeranyl
diphosphate synthase in the carotenoid biosynthetic pathway of N.
crassa (Nelson et al., 1989). Bts1p also contains five
conserved regions found in other FPP and GGPP synthases (Chen and
Poulter, 1994). These comparisons suggest that BTS1 encodes
GGPP synthase, an unidentified prenyltransferase of S.cerevisiae.
Disruption of the BTS1 Gene
To
investigate if BTS1 is required for the vegetative growth of
yeast cells, we disrupted one copy of this gene in diploid cells and
then performed tetrad analysis. This disruption was constructed by
exchanging a 0.65-kb segment of the coding region with the URA3 gene. The disrupted gene was then transformed into NY648 to
replace one of the chromosomal copies. The Ura transformants were selected, sporulated, and analyzed. In all of
the 48 tetrads examined, four viable spores were obtained. However, two
of the colonies in each of the tetrads displayed a growth defect at 25
°C. The small colonies were Ura
, indicating that
they contained the disrupted BTS1 gene. This was confirmed by
Southern blot analysis using BTS1 as a probe (see
``Materials and Methods''). Thus, BTS1 is not
essential for the vegetative growth of yeast cells. But in its absence,
growth is impaired. The growth of the disrupted strain (SFNY368 or
BTS1) was examined further at different temperatures. As
shown in Fig. 6, BTS1 cells (Fig. 6, a and d) grew as well as wild type at 30 °C (Fig. 6, b and c). However, at lower
temperatures (25 and 14 °C) a growth defect emerged. Only small
colonies appeared after 3 days at 25 °C (Fig. 6, a and d), while at 14 °C, the cells did not survive (Fig. 6, a and d). This result clearly
demonstrated that SFNY368 is cold sensitive for growth.
BTS1) is cold
sensitive for growth. Diploid cells with one copy of BTS1 disrupted were sporulated and subjected to tetrad analysis. In
each tetrad, two wild type spores (b and c) and two
spores containing the disrupted BTS1 gene (a and d) were germinated, purified, and grown at various
temperatures (14, 25, and 30 °C). The 25 and 30 °C plates were
incubated for 3 days, while the 14 °C plate was incubated for 7
days.
The BTS1 Gene Product Is Required for the Membrane
Attachment of Ypt1p and Sec4p
Ypt1p and Sec4p are two small
GTP-binding proteins that regulate intracellular membrane traffic
(Ferro-Novick and Novick, 1993). Like many small GTP-binding proteins,
they are synthesized in the cytosol but become membrane-bound to
perform their function (Rossi et al., 1991). The ability of
Ypt1p and Sec4p to bind to membranes is conferred by the addition of
the 20-carbon, geranylgeranyl moiety (Jiang et al., 1993). The
geranylgeranylation of these proteins is catalyzed by a protein
prenyltransferase that utilizes GGPP as a lipid donor. If BTS1 encodes GGPP synthase, disruption of this gene should result in
the depletion of GGPP. Consequently, the geranylgeranylation of Ypt1p
and Sec4p will be abolished. To test this hypothesis, we examined the
membrane association of these proteins in the BTS1 strain. SFNY368 was grown at 30 °C for 12 h until the A was 1.0 prior to shifting the cells to 14
°C for another 12 h. Aliquots of cells were removed at each time
point, converted to spheroplasts, lysed, and centrifuged at 450
g to remove unbroken cells and nuclei. Subsequently, these
lysates were centrifuged at 100,000 g for 1 h to
obtain supernatant and pellet fractions, and the distribution of Ypt1p
and Sec4p was examined in each of these fractions by Western blot
analysis. In wild type cells (Fig. 7, compare the amount in the
lysate (T) to the supernatant (S) and pellet (P)), most of Ypt1p and Sec4p was membrane-bound at both time
points, and the change in temperature did not affect their membrane
association (Fig. 7, compare 14 and 30 °C). However, in
SFNY368, most of the Ypt1p and Sec4p was soluble at both temperatures (Fig. 7, compare the amount in the lysate (T) to the
supernatant (S) and pellet (P)), although this defect
was more pronounced at 14 °C. Thus, the membrane association of
these small GTP-binding proteins is defective in BTS1 cells.
BTS1 cells. Cells were grown at 30 °C to exponential phase. 1
aliquot of cells was removed, pelleted, converted to spheroplasts, and
lysed. The remaining cells were shifted to 14 °C and grown for 12 h
before they were harvested and lysed. Lysates (T) were
centrifuged at 100,000 g to obtain pellet (P)
and supernatant (S) fractions. Samples were electrophoresed on
a 12.5% SDS-polyacrylamide gel and subjected to Western blot analysis
using anti-Ypt1p (A) or anti-Sec4p (B) antibodies. W.T., wild type.
Prenyltransferase Activity of Crude
Extracts
To test the hypothesis that BTS1 encodes
a geranylgeranyl diphosphate synthase, we cloned the gene into a pUC118
vector to express it in E. coli. Crude extracts of E. coli containing pUC118 (control) or pUC118/BTS1 were assayed
for prenyltransferase activity in the presence of
[1-C]IPP, using DMAPP or FPP as the allylic
substrate, and the reaction mixture was analyzed by HPLC. The
prenyltransferase activity observed was dependent upon the presence of
FPP, since no counts were obtained when the pUC118/BTS1 extract was assayed in the absence of FPP (not shown). The
radioactive product of this incubation co-eluted with unlabeled
synthetic GGPP, indicating that it is GGPP (Fig. 8). No
conversion of FPP to GGPP was seen with the pUC118 control. Both
extracts also showed low levels of activity in the conversion of DMAPP
to an acid-labile product. However, because the extent of conversion
was the same for both samples, this activity could not be due to Bts1p
(not shown). These findings confirm that BTS1 encodes a
geranylgeranyl diphosphate synthase.
C]IPP and FPP were injected onto an Asahipak
ODP-50 column, and 2-min fractions were collected. The symbol (x) indicates the background that resulted from a run in which
only unlabeled GGPP was injected.
bet2-1 Mutant Extracts Have a Lower Affinity
for GGPP
We next investigated the mechanism by which the
overexpression of BTS1 suppresses the lethality of the bet2-1 mutant. One possibility is that BTS1 suppresses by increasing the intracellular pool of GGPP, thereby
compensating for a mutant GGTase-II that has a lower affinity for GGPP.
To test this hypothesis, we measured the GGTase-II activity of wild
type and bet2-1 mutant extracts in the presence of
varying concentrations of GGPP. As a control, we also assessed the
activity of bet4-2 mutant extracts. BET4 encodes the -subunit of the GGTase-II (Li et al.,
1993; Jiang et al., 1993), and extracts prepared from this
mutant are devoid of GGTase-II activity. Unlike bet2-1,
the overexpression of BTS1 does not suppress the
temperature-sensitive growth defect of the bet4-2 mutant
(not shown). As shown in Fig. 9, the GGTase-II activity of the
wild type extract was saturated at 0.8 µM of GGPP. At
this concentration, the activity of the bet2-1 mutant
extract was approximately 5-10% of wild type. This activity was
significantly enhanced when the GGPP concentration was increased beyond
2 uM, and saturation was achieved at 6 µM. In
contrast, the GGTase-II activity of the bet4-2 mutant
extract could not be compensated for by increasing the concentration of
GGPP. The calculated K
values of
GGTase-II in the bet2-1 mutant and wild type were
3.6 and 0.4 µM, respectively. Therefore, it appears
that GGTase-II in the bet2-1 mutant has a reduced
affinity for GGPP, which results in a decrease in prenylation activity.
By increasing the amount of GGPP that is added to the assay,
prenylation activity is efficiently restored. This result provides a
clear explanation for the suppression of bet2-1 by BTS1.
H]GGPP using wild type (W.T.) (open
circle), bet2-1 (solid circle), and bet4-2 (open triangle) mutant extracts. Assays
were performed at 30 °C for 30 min with 25 µg of yeast extract
and the indicated concentration of [H]GGPP. Each
value is an average of duplicate
determinations.
-subunit of this enzyme complex, forms
a complex with Bet4p, the -subunit (Jiang et al., 1993).
Mrs6p is an escort protein that presents protein substrate to the
Bet2p-Bet4p complex (Jiang et al., 1994). During
geranylgeranylation, the Bet2p-Bet4p complex binds to and transfers
GGPP to Ypt1p, Sec4p, and other small GTP-binding proteins. In an
attempt to identify new genes whose products may interact with Bet2p,
we isolated a suppressor of the bet2-1 mutant. Our data
demonstrates that this suppressor gene, called BTS1, encodes a
geranylgeranyl diphosphate synthase, an unidentified prenyltransferase
of the yeast isoprenoid biosynthetic pathway. The BTS1 gene
product functions on this pathway to convert FPP to GGPP.) for
GGPP, which is compensated for by higher concentrations of GGPP, this
alternate possibility is most likely. According to this model,
additional copies of BTS1 should result in higher
intracellular concentrations of GGPP and enhanced suppression of bet2-1, thus explaining why the suppression of bet2-1 by BTS1 is gene dosage dependent.
BTS1 strain was only cold sensitive for growth. Furthermore, the growth
of this strain was not impaired at 30 °C or higher temperatures.
When the membrane association of Ypt1p and Sec4p was examined in
BTS1 cells grown at 30 °C, a small fraction of each
of these proteins was membrane bound. Thus, BTS1-depleted
cells are able to prenylate proteins at a level that is sufficient to
sustain cell growth at higher temperatures. When these cells were
shifted to 14 °C, less membrane-bound Ypt1p and Sec4p was detected,
implying that growth ceases as a consequence of the failure to
prenylate these essential proteins.BTS1 cells do not support the
transfer of [H]FPP onto Ypt1p. Thus, it is more
likely that another prenyltransferase, such as hexaprenyl diphosphate
synthase, might produce small amounts of GGPP as an intermediate
product during the elongation of FPP to longer polyisoprenoid chains.
In the BTS1 strain, GGPP may be formed in this way,
enabling yeast cells to survive at certain temperatures in the absence
of the geranylgeranyl synthase.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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