(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
-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.
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) ()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).
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 g.
Samples were then subjected to Western blot analysis using anti-Ypt1p
and anti-Sec4p antibodies.
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 (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.
Figure 7:
The
membrane attachment of Ypt1p and Sec4p is defective in 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.
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-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.
Figure 9:
Saturation curves for
[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.
Previously, we have shown that the yeast GGTase-II is
composed of three subunits (BET2, BET4, and MRS6). Bet2p, the -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.
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) 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.
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 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.
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 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.