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(Received for publication, June 1, 1995; and in revised form, August 18, 1995) From the
In Saccharomyces cerevisiae, the mating
pheromone-initiated signal is transduced by a heterotrimeric G protein
and normally results in transient cell cycle arrest and
differentiation. A null allele of the G
The budding yeast Saccharomyces cerevisiae has two
haploid cell types, a and Mating between
haploids of opposite mating type is initiated by the binding of
pheromone, secreted from each cell type, to specific receptors
expressed on cells of the opposite mating type. Pheromone binding
triggers G Mating signal transduction (reviewed in (3, 4, 5) ) begins with the binding of
pheromone to its cognate receptor, Many components of the arrest-differentiation pathway
downstream of G In this study we describe the isolation of
three high copy gpa1 suppressors. Two of these are known to
control the expression of components of this pathway. The third is a
novel gene with truncated forms that can suppress either cell cycle
arrest alone or both cell cycle arrest and differentiation. We
demonstrate that this suppressor protein is tightly associated with the
plasma membrane and that it acts to block G protein signaling at the
level of G
Suppressors of gpa1 lethality were
isolated by first transforming GU1 cells with a library of yeast
genomic fragments cloned into pUV1, a 2-µ based plasmid carrying
the URA3 marker (generous gift of Junichi Nikawa and Michael
Wigler). 1% of the transformed cells were plated on SC-uracil to
determine the total number of transformants. The remainder were grown
in 20 ml of SC-uracil media for 2 days, and a sample of these cells
(200 µl) was plated on SC-uracil-arginine with canavanine (60
µg/ml) added. Individual colonies were picked and grown in
SC-uracil for preparation and analysis of plasmid DNA.
The SYG1 disruption construct using the LEU2 marker was made as follows. The KpnI/XbaI SYG1-1 fragment was transferred
into pBluescript KS (pKS, Stratagene) to form pKS-SYG1-1. To introduce
the LEU2 marker, the HindIII/EcoRV fragment
of SYG1-1 was removed from pKS-SYG1-1 and replaced by LEU2 on a HindIII/SmaI fragment. A NotI/SmaI syg1::LEU2 disruption
fragment was used to transform strains SP1 and FY250. Southern and
Northern analyses were used to confirm syg1::LEU2 disruptions. Full-length pUV1-SYG1 and the deletion pUV1-SYG1 Three COOH-terminal
deletion constructs were made using PCR-generated fragments.
pUV1-SYG1 pADNS-SYG1-1 was
constructed by first moving the HindIII/NotI fragment
of pUV1-SYG1-1N into pADNS (16) . The HindIII site was
changed to a SalI site using the adaptor 5`-AGCTGTCGAC-3`.
This construct was designated pADNS-SYG1-1SN. The most
NH The NH YEp-
Halo assays were performed essentially as described (21) except that the experimental strains were cultured as for
the quantitative mating assay and approximately 5 FUS1 induction analyses were done using
the bar1/sst1 strain GPY74-15Ca which was
treated with STE4
The S. cerevisiae cDNA library used was created by Jeff
Kuret (Cold Spring Harbor Laboratory)(28) . Standard screening
and hybridization conditions were used. Sequencing was performed using
dideoxy termination reactions. Data base searches were performed
using the BLAST algorithm(29) .
A total of seven
independent plasmids that could suppress gpa1 were isolated.
Following restriction mapping, cross-hybridization and sequence
analysis, it was determined that five clones contained the MAT The remaining clone was found to contain a portion of a novel gene, SYG1 (suppressor of yeast gpa1). The cloned form of
the gene, SYG1-1, was also capable of suppressing gpa1 in an Sequence analysis revealed that SYG1-1 had a 417-amino acid
open reading frame that is a carboxyl-terminal truncation of SYG1 (Fig. 1). A portion of this sequence was used as a probe to
isolate the remainder of the SYG1 coding region from a S.
cerevisiae cDNA library. Four distinct SYG1 cDNAs were
isolated and sequenced. All were collinear with the original SYG1-1 clone and contained additional 3` sequences that extended the open
reading frame to 902 amino acids (Fig. 1B). The cDNA
sequences were used to reconstruct a full-length SYG1 coding
region attached to the SYG1 promoter. Full-length SYG1, expressed from the same high copy expression plasmid as SYG1-1, was an extremely weak suppressor of gpa1.
Relative to SYG1-1, suppression by SYG1 resulted in
fewer colonies (Fig. 2), and these took twice as long to appear.
Figure 1:
A, restriction
map of SYG1. The SYG1 clone is shown as a 3-kb
fragment extending from the upstream genomic XbaI site to the
end of the SYG1A cDNA. The hatched box represents the
open reading frame. Restriction sites are labeled as follows: X, XbaI; H, HindIII; P, PstI; N, NheI; RV, EcoRV; S, SacI, and RI, EcoRI. B,
predicted amino acid sequence of SYG1. The termination codon
is marked by a dot. The filled diamond positioned
above the sequence indicates the end of the SYG1 sequence
obtained in the original SYG1-1 clone. Delta symbols above the sequence indicate the positions of the deletion
constructs tested for gpa1 suppression. Predicted
transmembrane domains are underlined. SYG1 was
recently determined to be located on chromosome IX (accession number
Z46861). C, alignment of two regions of predicted protein
sequence for Syg1p and four related yeast sequences; N2052, J0336,
YCR524, and Pho81p (accession numbers X77395, X77688, X56909, and
X52482, respectively). Identical residues are boxed, conserved
residues (
Figure 2:
Suppression of gpa1 cells by SYG1 constructs. MAT
A striking feature of the SYG1 primary sequence was
revealed by hydropathy and hydrophobic moment analyses. As shown ( Fig. 1and 3), several distinct hydrophobic domains are located
within the full-length protein, and eight of these are predicted to
span the membrane. The eighth domain was noted to be amphophilic,
raising the possibility that it may form a pore. The truncation giving
rise to SYG1-1 resulted in disruption of the first hydrophobic
domain and deletion of the others. This suggested that removal of these
domains conferred upon SYG1-1 its potent gpa1 suppression activity. Northern analysis (Fig. 4) showed
that SYG1 is expressed as a 2.8-kb transcript in both a and
Figure 4:
Northern blot analysis of SYG1 expression. Lane 1, GU1 MATa; lane 2, GU2
MAT
Strains that carry a syg1::LEU2 mutation showed no
noticeable change in mating efficiency. These cells did not display any
generalized defect in cell growth under normal conditions nor when
exposed to stresses including growth at 37 °C, heat shock treatment
at 55 °C, and hyperosmotic media. They were able to switch to
alternate carbon sources, undergo meiosis, and give rise to viable
spores. We considered the possible existence of genes that are
functionally redundant with SYG1 and can thereby mask the
effects of the syg1 null mutation. Although we have not
detected any related sequences using low stringency hybridizations,
four sequences with significant, though limited, similarity to SYG1 were identified through a data base search (Fig. 1C). The two regions of similarity with SYG1 lie within the sequence of SYG1-1. In addition, three of
these sequences (N2052, J0336, and YCR524) encode predicted proteins
that, like SYG1, have amino-terminal spans of about 400
hydrophilic residues followed by multiple strongly hydrophobic domains.
Overexpression of the amino termini of these proteins does not confer
any detectable gpa1 suppression, however (data not shown). We performed a carboxyl-terminal deletion analysis of SYG1 to further examine structure-function relationships (Fig. 5). SYG1
Figure 5:
Deletion mutants of SYG1. All
constructs have a wild type amino terminus and are deleted from the
carboxyl terminus. The deletion number indicates the extent of SYG1 amino acid sequence present. The
Figure 6:
Proliferation of differentiated cells. GU1
cells were transformed with pUV1-SYG1-1 and pUV1-SYG1
Figure 7:
A, subcellular localization of
Syg1
This subcellular
localization result suggested that Syg1
To address the
possibility that SYG1-1 was interfering with signal
transduction by directly or indirectly altering expression of signaling
components, we performed quantitative RT-PCR. High copy SYG1-1 expressed in the wild type strains SP1 and FY250, as well as in
the gpa1 pUV1-SYG1-1 strains GU1S and GU2S, had no effect on
mating type-specific expression of pheromone receptors (in a MATa background STE2 is expressed at normal
levels and STE3 is not expressed, as in wild type cells) nor
on the normal abundance of STE4 message (data not shown). In
addition, overexpression of SYG1-1 did not appear to block the
induction of FUS1 after pheromone treatment. This result was
confirmed using direct detection of a FUS1 promoter-driven LacZ construct(23) . As seen in Table 2,
expression of SYG1-1 only slightly dampens FUS1 induction. As expected, expression of MAT
The ability of SYG1 and SYG1-1 to modulate response to pheromone was tested
using a halo assay. Application of pheromone was used to create a zone
of growth inhibition that reflects the normal cell cycle arrest
response following activation of the mating pathway. Although wild type
cells with SYG1 gave normal halos, cells overexpressing SYG1-1 gave rise to halos that became partially filled with
colonies (Fig. 8A). Colonies within the halo were
visible soon after those that formed the lawn. A similar result was
seen when GPA1 was overexpressed in wild type cells (Fig. 8B), probably due to the sequestering of Ste4p
into an inactive complex with Gpa1p. In both cases, the diameter of the
halo, representing the initial sensitivity to pheromone, was unchanged
from wild type cells. These data are consistent with the FUS1-lacZ data in which SYG1-1 had little
effect on initial FUS1 induction. Simultaneous overexpression
of GPA1 and SYG1-1 did not further enhance the growth
of cells within the halo (Fig. 8B). This indicated that
Gpa1p and Syg1-1p may compete for the same target (Ste4p). It also
suggested that there may be a limit to the level of signal repression
and/or adaptation enhancement that can be achieved in this way.
Figure 8:
Halo assays. The strains used were SP1 (wt), GPY74-15Ca (sst1), YDM400 (sst2), SP1-SN (syg1), GU1 (gpa1 with pLGC)
and GU1S (gpa1 with pSYG1-1). pSYG1-1 and pSYG1 designate that expression constructs pUV1-SYG1-1 and pUV1-SYG1,
respectively, were transformed into these strains. 20 µg of
The
turbid halo results indicated that SYG1-1 may function to
relieve cell cycle arrest by stimulating adaptation. Sst1p and Sst2p
mediate separate adaptation pathways which, when disrupted (sst1 or sst2), result in a supersensitive response to
pheromone. The Bar1p/Sst1p protease normally contributes to adaptation
by degrading For GPA1, the co-overexpression of full-length SYG1 did not alter the halo fill-in effect (Fig. 8B). Similarly, overexpression of non-suppressing
truncations of SYG1 did not modulate pheromone recovery
mediated by SYG1-1 (data not shown). In addition, syg1::LEU2 mutants showed no alteration in halo formation from
wild type cells (Fig. 8E). In cells expressing SYG1-1 that are also gpa1 no halo is ever visible (Fig. 8G). Expression of SYG1-1 had very
little effect on mating efficiency. Both wild type cells (SP1) and GU1
cells transformed with pUV1-SYG1-1 mated at levels that were
essentially unchanged from untransformed controls (within 2-fold, data
not shown). gpa1 cells that carried the SYG1-1 suppressor did show reduced mating efficiency (about 20-fold, data
not shown). Given that these cells showed no pheromone response in a
halo assay, it was surprising that they mated at this level of
efficiency (no mating with cells of the same mating type was observed).
This is, however, similar to the level of mating seen in gpa1
Figure 9:
Suppression of STE4 overexpression. FY251 cells carrying the galactose inducible STE4 plasmid (YCpGAL-STE4) were transformed with plasmids
expressing the indicated genes (vector = pUV2, GPA1 = pUG, SYG1-1 = pUV1-SYG1-1, SYG1 = pUV1-SYG1). Four independent colonies were patched onto
sucrose-containing media and replica plated to sucrose or
galactose-containing media.
To test whether Syg1-1p might be suppressing
through a direct interaction with Ste4p, as is the case for Gpa1p, we
examined the ability of this allele to suppress STE4
Figure 10:
Suppression of STE4
Figure 11:
Interaction between Syg1
The interaction between Syg1
We have shown that the gpa1 mutation, which leads to
constitutive G Other cases
of high copy or dominant suppressors of gpa1 have been
reported, and all seem to function downstream of G We have described the isolation and characterization of SYG1-1 (a truncated form of the SYG1 gene) that is a novel high
copy suppressor of gpa1. SYG1 itself does not have expression
characteristics common to many genes directly involved in the mating
pathway; its transcription is not induced by pheromone nor is it
restricted to haploids. Although SYG1 may not normally be
involved in the pheromone response pathway, we present data consistent
with its participation in G protein-mediated signaling. The ability
of SYG1-1 and SYG1 Despite lacking predicted transmembrane domains and a signal
sequence, Syg1 In mammalian cells, as in
yeast, G We have demonstrated that in otherwise wild type cells,
high level expression of SYG1-1 stimulates recovery from
pheromone, as judged by halo assay. Many factors required for
adaptation and resumption of vegetative growth have been identified.
These include pheromone receptors(21, 52) ,
Sst2p(21, 31, 32) , and Ste4p(53) .
In addition, mutant forms of Ste4p that are impaired for transducing
the mating signal still retain their ability to stimulate
adaptation(54) . The binding of Syg1-1p may promote pheromone
recovery not only by sequestering Ste4p but also by accentuating its
adaptation function. Alternatively, high levels of Syg1-1p may directly
activate this, or an as yet unidentified, adaptation pathway. Furthermore, as with GPA1 overexpression, SYG1-1 overexpression appears to dampen but not block the mating signal
cascade in wild type cells (pheromone recovery and mating results).
There are other examples of potent constitutive signaling mutation
suppressors which do not block normal signaling in wild type cells. For
example, high copy suppressors of activated mutant RAS2 One striking outcome of the SYG1 deletion analysis was the ability of SYG1 The ability of Syg1-1p to suppress constitutive pheromone signaling,
stimulate adaptation in wild type cells, and physically interact with
Ste4p suggests that Syg1p may normally respond to or transduce a signal
via G
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 25435-25444
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Deficiency by Interacting with the
Subunit (*)
ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(GPA1/SCG1) subunit results in cell death due to
unchecked signaling from the G
(STE4, STE18, respectively) heterodimer. We have identified three
high copy suppressors of gpa1 lethality. Two of these genes
encode known transcription factors, Mat
2p and Mcm1p. The third is
a truncated form of a novel gene, SYG1. Overexpressed wild
type SYG1 is a weak suppressor of gpa1. In contrast,
the isolated mutant allele SYG1-1 is a strong suppressor that
completely blocks the cell cycle arrest and differentiation phenotypes
of gpa1 cells of both mating types. One deletion mutant (SYG1
340) can suppress the cell cycle arrest associated
with gpa1, but the cells retain a differentiated morphology. SYG1-1 can suppress the effects of overexpressed wild type
G but is not able to suppress the lethality of an activated G
mutant (STE4). Consistent with these
genetic observations, the suppressing form of Syg1p can interact with
the STE4 gene product, as determined by a two-hybrid assay. SYG1-1 is also capable of promoting pheromone recovery in wild
type cells, as judged by halo assay. The sequence of SYG1 predicts eight membrane-spanning domains. Deletion mutants of SYG1 indicate that complete gpa1 suppression requires
removal of all of these hydrophobic regions. Interestingly, this
truncated protein localizes to the same plasma membrane-enriched
subcellular fraction as does full-length Syg1p. Three hypothetical
yeast proteins, identified by their similarity to the SYG1 primary sequence within the gpa1 suppression domain, also
appear to have related structures. The properties of Syg1p are
consistent with those of a transmembrane signaling component that can
respond to, or transduce signals through, G
or G.
, which are defined by the gene
cassette expressed from the MAT locus. Haploids of opposite
mating type can conjugate to yield a/ diploids. Expression
of the genes that determine cell identity (a, , or a/) is controlled in large part through the transcription
factor encoding genes MATa1, MAT1, MAT2, and MCM1 (reviewed in (1) and (2) ).
cell cycle arrest, a differentiation program
that leads to morphologically altered cells (shmoos) that are enlarged
and elongated, and induction of various genes required to consummate
cell fusion (reviewed in (3) ). In this state, cells of
opposite mating type fuse at their projection points. Subsequent
nuclear fusion leads to mating resolution and eventual resumption of
mitotic growth. Cell cycle arrest and differentiation can also be
brought about by exogenously supplying the appropriate pheromone in the
absence of opposite mating type cells. After some time, however, the
arrested cells adapt to the pheromone and resume vegetative growth. -factor receptor (Ste2p) on a cells or a-factor receptor (Ste3p) on cells.
Pheromone binding triggers activation of a heterotrimeric G protein by
releasing the G subunit (encoded by the GPA1 gene, also
called SCG1) from the signal transducing G subunits
(encoded by the STE4 and STE18 genes, respectively).
Even in the absence of pheromone, cell cycle arrest, differentiation,
and ultimately cell death can result from (i) a deficiency of the
G
subunit(6, 7) , (ii) a G subunit mutant
that is insensitive to repression by G(8, 9) , or
(iii) overexpression of the wild type G subunit(10) .
These data demonstrate that G is the principal transducer of
the signal for cell cycle arrest and differentiation and that G
acts as a repressor that is necessary for signal attenuation. It is
unclear, however, whether G works solely through G
repression or if it can actively stimulate an independent adaptation
pathway.
have been identified through the analysis of
recessive mutants, many of which are sterile (ste). These
include the protein kinases Ste20p, Ste7p, Ste11p, Fus3p, and Kss1p.
Cell cycle arrest is potentiated by Far1p through a direct interaction
with a cyclin-dependent kinase(11) , while pervasive changes in
gene expression are mediated by the action of the Ste12p transcription
factor(12) . Less is known, however, about the immediate
effector of G
.
.
Strains, Transformation, Selection, and Propagation
Procedures
Strains are described in Table 1. Strains SP1
and RS22-6C were mated to make diploid strain SR. Strain SRgHU
was created by sequentially transforming strain SR with a gpa1::HIS3 EcoRI fragment from pgH and a gpa1::URA3
EcoRI fragment from pgU in order to consecutively eliminate both
copies of GPA1. Strain SRgHU was then transformed with the
pLGC plasmid. Following sporulation and tetrad dissection, strains GU1
and GU2 were obtained. Strain DB6 was generously provided by Duane
Jenness, University of Massachusetts, Worcester. Strain DBC was created
by first plating DB6 cultures on canavanine-containing media to select
for can1 mutants. After introducing pU2C, transformants
were screened on the basis of colony color and a Leu phenotype for isolates which had lost pDJ117. YDM400 was
generously provided by Doreen Ma and Jeremy Thorner, University of
California, Berkeley. GPY74-15Ca was generously provided by Greg
Payne, UCLA. Standard formulations were used for YPD, synthetic
complete (SC), (
)and sporulation media, and all yeast
cultures were grown at 30 °C. All yeast transformations were done
as described previously(13) , and all plasmids were propagated
in DH5 cells.
Plasmids
The GPA1 knock out constructs
(pgH and pgU) were created by inserting blunt-ended HIS3 and URA3 fragments between the internal SspI sites of GPA1. pLGC was created by inserting the Klenow-blunted EcoRI fragment of GPA1 into the PvuII site
of pTLC(14) , which carries the LEU2 and CAN1 genes. pUV1-SYG1-1 is the library-derived clone. Vector pUV2
differs from pUV1 only in the orientation of URA3. pU2C
was created by inserting a 4-kb CAN1 gene fragment (from
pYeCAN1-2, provided by Kelly Tatchell, North Carolina State
University) between the KpnI and SacI sites of
pUV1-2, the library isolate of MAT 2. pUG carries
the GPA1 gene on a 2-µ based URA3 vector. pUGC is
a version of pUG that includes the CAN1 gene. The
p416-SYG1-1 centromeric version of SYG1-1 was created by
cloning the KpnI/XbaI SYG1-1 fragment from
pUV1-SYG1-1 into the KpnI/XbaI sites of pRS416 (15) .554 were
created using the cDNA clones pKS-SYG1A and pKS-SYG1D, respectively.
For both constructs, the polylinker EcoRI site of pUV1-SYG1-1
was changed to a NotI site using the adaptor
5`-AATTGCGGCCGC-3` (all oligos were obtained from Integrated DNA
Technologies) to form pUV1-SYG1-1N. To make pUV1-SYG1, the polylinker XhoI site of pKS-SYG1A was changed to a NotI site
using the oligomeric adapter 5`-TCGAAGCGGCCGC-3` to make pKS-SYG1AN.
pKS-SYG1D already contained a polylinker NotI site at the 3`
end of the clone. The 3`-SYG1 NheI/NotI fragments
from pKS-SYG1AN and pKS-SYG1D were used to replace the NheI/NotI fragment of pUV1-SYG1-1N. The construct
pUV1-SYG1 contains its own natural stop codon. A second pUV1-SYG1
construct was created from an independent cDNA isolate, and the two
pUV1-SYG1 constructs behaved identically.519, pUV1-SYG1464, and pUV1-SYG1400 were
created by replacing the SacI/EcoRI fragment of
pUV1-SYG1554 with PCR-generated fragments whose SYG1 sequence ended at the indicated codon and introduced an EcoRI site for cloning purposes. For pUV1-SYG1464 and
pUV1-SYG1400, a TAA stop codon was introduced after the last SYG1 codon, whereas for pUV1-SYG1519, the TAG stop codon
was supplied by the vector. Each PCR used the upstream primer YS5
(5`-TTCATGTCGTACGCCAGG-3`) which is 5` of the SacI site. The
downstream primers were 5`-TAGAATTCTTAAATCGATCTGTTGTTTCT-3`
(SYG1400), 5`-TAGAATTCTTAGGTTCTATGCCAGATAAA-3` (SYG1464), and
5`-TAGAATTCTTATCCAAAGCGAAACTTAAC-3` (SYG1519). For cloning, the
PCR-generated fragments were digested with SacI and EcoRI prior to gel purification and ligation. PCRs were done
using three cycles of (94 °C for 1 min, 40 °C for 1 min, 72
°C for 1.5 min) followed by 25 cycles of (93 °C for 0.5 min, 55
°C for 0.5 min, 72 °C for 2 min) followed by 72 °C for 5
min. Multiple independent PCR-derived clones of each construct were
assayed with the same result. pUV1-SYG1340 was constructed by
removing the 5`-SacI restriction fragment from pUV1-SYG1-1
using the internal SacI site and a polylinker SacI
site. pUV1-SYG1199 was constructed by removing the
5`-NheI/NotI fragment from pUV1-SYG1-1N by digesting
with NotI and NheI, blunting the termini with Klenow,
and then ligating. For pUV1-SYG1340 and pUV1-SYG1199, the
stop codon signal was provided by vector sequence.
-terminal portion of SYG1-1 was moved as a
PCR-amplified fragment which introduced a SalI site and an
in-frame start ATG codon. The NH-terminal PCR primer was
5`-AGTCGTCGACAATGAAGTTTGCTGACCAT-3`. The downstream amplification
primer 5`-GCTGTCGATGCTATTGAC-3` was positioned 3` of the SYG1-1
PstI site. The amplified fragment was digested with SalI
and PstI prior to being ligated into pADNS-SYG1-1SN. The
PCR-amplified portion of pADNS-SYG1-1 was sequenced to confirm that no
errors had been introduced. This construct was tested and shown to have gpa1 suppression activity in LG1TG and LG2TG, gpa1 strains that carry the leu2 mutation, although the level
of suppression (number of survivors on canavanine-containing media) was
a few fold below what is seen for pUV1-SYG1-1 in GU1 or GU2 cells.
pADNS-SYG1 was constructed by substituting the SacI/NotI fragment of cDNA clone pKS-SYG1E for the
incomplete pADNS-SYG1-1 SacI/NotI fragment. pKS-SYG1E
differs from pKS-SYG1A only in that pKS-SYG1E contains a poly(A) tail.
To create the COOH-terminal hemagglutinin (HA)-tagged expression
constructs, the SalI/NotI fragment from pADNS-SYG1
was cloned into pADCLX, which was derived from pAD54 (13) and
has an in-frame NotI site preceding, and a unique KpnI site following, the HA epitope. This intermediate
construct was called pCLXSF. To create pADCLX-SYG1400, the SacI/NotI fragment of pCLXSF was replaced by the PCR
fragment generated using YS5 as the upstream primer and
5`-GCTAGCGGCCGCCCAATCGATCTGTTGTTTCT-3` as the downstream primer. To
create pADCLX-SYG1, a fragment with a 3` in-frame NotI site
was generated by PCR using the upstream primer 5`-TTGTGCGGTCTGTTCCAT-3`
and the downstream primer 5`-GCTAGCGGCCGCCCCATAATACTTTCCACTTC-3`. This
PCR product was cloned as an EcoRI/NotI fragment into
pUV1-SYG1 and then moved as a SacI/NotI fragment into
pCLXSF. To create pCLA-SYG1400, the expression construct which
fused the LexA coding sequence to the COOH terminus of SYG1400, the following steps were taken. The vector pKSN
was made by changing the XhoI site in the vector pKS to an NcoI site using the adaptor 5`-TCGACCATGG-3`. LexA was PCR amplified using the upstream primer
5`-GCATGCGGCCGCGGATCCTTATGAAAGCGTTAACGGCC-3` which introduced in-frame NotI and BamHI restriction sites and the downstream
primer 5`-TACGCCATGGTTACAGCCAGTCGCCGTT-3` which added a stop codon and NcoI restriction site. The resulting LexA amplicon
was cloned into pKSN. From pKSN-LexA, a NotI/KpnI LexA fragment was used to replace the HA epitope of
p9CL-SYG1400, creating pCLA-SYG1400. p9CL-SYG1400 has
the SphI/SphI fragment of pADCLX-SYG1400
inserted into the SphI/SphI backbone of
pGBT9(17) .-terminal Gal4(768-881)
activation domain fusion expression vector pKB40.1 and the hybrid
constructs pGAD2F.N, pMA424, pKB33.23, and pKB24.5 (18) were
the generous gift of M. Whiteway (National Research Council, Montreal).
The NH-terminal LexA fusion expression vector pBMT116-GPA1
was made by moving the EcoRI/SalI fragment of
pKB33.23 into pBMT116. ()Similarly, the
NH-terminal, Gal4(768-881) activation domain fusion
expression vector pGAD424-STE18 was made by moving the EcoRI/SalI fragment of pKB24.5 into pGAD424 (17) .2 was constructed by ligating the SacI/SmaI fragment of the original MAT2 genomic isolate into the SacI/SmaI sites of YEp13(14) . YEp-MCM1 was
constructed by ligating the SacI fragment of the original MCM1 genomic isolate into YEp13. YCpGAL-STE4 for
overexpressing STE4 under galactose-inducing conditions was
generously provided by Steven Reed, Scripps Research Clinic.Cell Extract Preparation, Fractionation, Membrane
Stripping, and Immunoblot Analysis
Crude cell extract
preparation was done as described (13) except that the lysis
buffer was made of 20 mM 2-MOPS, pH 6.0, 1 mM
MgCl, 100 µM EDTA, and 1 mM dithiothreitol. Crude cell extract fractionation by differential
centrifugation was done as described (19) . Immunoblot analysis
using anti-HA monoclonal antibody was done as described(13) .
Marker proteins were detected similarly using the following polyclonal
anitbodies: anti-Na-K
-ATPase (1:500
dilution) for the plasma membrane, anti-glucose-6-phosphate
dehydrogenase (1:3,000 dilution) for the cytosol, and anti-Sec63p
(1:2,500 dilution) for the endoplasmic reticulum. These antibodies were
generous gifts from the laboratories of Nathan Nelson (Roche Institute,
Nutley, NJ), Greg Payne (UCLA), and David Meyer (UCLA), respectively.
Immunoblots treated with polyclonal primary antibody were incubated
with alkaline phosphatase-conjugated goat anti-rabbit antibody
(Bio-Rad, 1:2,500 dilution). For the membrane stripping experiments,
aliquots of the 10,000
g pellets were diluted into 0.5
ml of stripping buffer (0.5 M NaCl, 2 M urea, 10
mM Tris-HCl, pH 10.5, 0.5% -mercaptoethanol, 0.5% Triton
X-100, or 1% SDS) and incubated for at least 1 h on ice with occasional
mixing. The samples were then centrifuged at 10,000 g for 10 min at 4 °C. The pellets were resuspended in lysis
buffer, and the supernatants were either concentrated at 4 °C using
Microcon-10 microconcentrators (Amicon) or acetone-precipitated.Mating, Halo, Two-hybrid, STE4
Quantitative
mating assays were conducted essentially as described (20) except that approximately 10, and
Overexpressed Wild Type STE4 Suppression Assays
cells of the
tester strains DC14 or DC17 were used, and the experimental strains
were cultured in plasmid maintenance synthetic medium before being
switched into YPD medium. Mating efficiencies were calculated as the
ratio of diploid colonies formed to the titer of experimental haploid
cells used. 10
cells were inoculated into either SC or YPD soft agar which was
poured onto the corresponding media. Results shown used the YPD
conditions which facilitated a slightly enhanced response to
-factor over the synthetic medium conditions. Synthetic
-factor (Sigma) was added to the filter discs in a volume of 10
µl of water.-factor at a concentration of 1 µg/ml. After 2 h
of incubation, >80% of the cells were unbudded. Time points for RNA
analyses were taken prior to addition of pheromone (T =
0) and 15, 60, 120, and 180 min after addition of pheromone. FUS1 induction as determined by reverse transcribed-PCR occurred as
described(22) . FUS1 induction was also measured from
a 2-µ based FUS1-LacZ reporter construct, pSB234 (23) . This was transformed into GPY74-15Ca cells,
followed by pADNS, pADNS-SYG1-1, YEp-2, and YEp-MCM1. Cells were
grown overnight in selective media, switched to YPD, treated with
-factor for 1 h, lysed, and assayed for -galactosidase
activity(24) . The two-hybrid -galactosidase liquid assays
were done similarly except that lysis was accomplished by two cycles of
freeze-thaw using liquid nitrogen. To test for His complementation, individual transformants were patched onto media
selecting for each of the two introduced plasmids and then replica
plated to media that also selected for HIS3 expression. The
patches on His
selection media were replica plated a
second time onto the same media to eliminate background growth. Filter
lift
-galactosidase assays were done as described(25) . suppression was assayed by transforming
DBC cells, culturing them for 1-2 days in SC-leucine media, and
then plating 1-10 µl of culture onto
SC-leucine-arginine+canavanine media to select for loss of the
pU
2C maintenance plasmid. Suppression of overexpressed wild type STE4 was done essentially as described (10) except
that individual transformants were first patched onto media containing
sucrose and subsequently replica plated onto media containing galactose
(3%). To enhance the growth difference, these were replica plated onto
the same media a second time.RNA Analysis, Hybridizations, Phage Library Screening,
Sequencing, and PCR
Total RNA was prepared using the hot phenol
method(26) . For Northern analysis, total RNA was fractionated
on a 0.8% agarose, 2% formaldehyde gel prior to transfer to reinforced
nitrocellulose. Reverse transcribed-PCR studies employed conditions as
described (27) except that 2 µg of total RNA was used for
cDNA synthesis and an annealing temperature of 54 °C was used for
all PCR reactions. The number of PCR amplification cycles and agarose
percentage required for fragment detection by ethidium-stained agarose
gel analysis depended on the fragment. The upstream and downstream
primers, respectively, and the fragment sizes were as follows: ACT1 (5`-TTGAGTGAACGTGGTTAC-3`, 5`-TTGTGGTGAACGATAGAT-3`, 500 bp); STE2 (5`-GATGCTGACGTCGATATC-3`, 5`-TTCTGGCTTCCTCATCAG-3`, 750
bp); STE3 (5`-CGTCAAGGACCTTGTGAT-3`, 5`-CTGGAGAGTAGCAAAGAC-3`,
1000 bp); STE4 (5`-ATCAGATGGACTCGATAA-3`,
5`-GAGACCATATTTTCATGG-3`, 1300 bp); and FUS1 (5`-CACCAGTTGATAATACCC-3`, 5`-TTCTTGGAGACAGTCACC3-`, 375 bp).
Isolation of gpa1 Suppressors
To identify genes
involved in regulating the pheromone response in S.
cerevisiae, a genetic selection for yeast sequences capable of
rescuing gpa1 cells was undertaken using a plasmid exchange
protocol. Strain GU1 has a gpa1 null allele but is viable
because it has an extrachromosomal copy of GPA1 carried on the
plasmid pLGC, which also encodes a positive selectable marker (LEU2) and a negative selectable marker (CAN1). GU1
cells were transformed with a library of yeast genomic fragments
carried on a 2-µ based high copy plasmid. Approximately 5
10 transformants were obtained from seven separate
transformations. These cells were grown in synthetic media which
selected for the library plasmid but allowed for the loss of pLGC.
Transformants which lost the pLGC plasmid were identified using the
negative selectable marker. Plasmid DNA was isolated from individual
colonies and used to transform fresh GU1 cells which were then tested
for suppression of gpa1 lethality.2 gene and one clone carried MCM1.
When tested in cells (GU2), MAT2, but not MCM1, was able to give gpa1 suppression. These genes
encode proteins that act together and with other factors to control
transcription of mating type-specific genes, including some that are
required for the pheromone signaling pathway(1, 2) . strain (GU2) that is otherwise isogenic with GU1. This
demonstrated that suppression is likely to involve a generalized block
of cell cycle arrest and not a mating type-specific interference.
0.3 from Dayhoff table(60) ) are shown, and amino
acid positions are given at the right. There are no gaps
introduced.
gpa1 pLGC cells
(GU2) were transformed with the indicated high copy expression plasmid (vector = pUV2, SYG1-1 = pUV1-SYG1-1, SYG1340 = pUV1-SYG1340, and SYG1 = pUV1-SYG1), grown, and plated on selection plates as
described under ``Materials and Methods.'' Growth requires
the loss of the pLGC plasmid and suppression of gpa1.
Approximately equal numbers of cells were
plated.
haploids as well as in a/ diploids.
Expression in GU1 and GU2 cells which overexpress GPA1 (lanes 1 and 2), appeared to be slightly
repressed compared to levels in a wild type haploid cell (lane
4) or a diploid cell (lane 3), suggesting possible
regulation by some component(s) of this pathway. Two smaller
transcripts also appeared on the blot. These are likely to be SYG1 derived since they were eliminated in a syg1 deletion
strain (lane 5). No change in SYG1 expression level
was seen following treatment with -factor (data not shown).
; lane 3, SRgHU diploid; lane 4, SP1 MATa; lane 5, SP1-SN Mata syg1::LEU2. Indicated to the left of the blot are the sizes of the RNA molecular weight standards
(Life Technologies Inc., 0.24-9.5-kb RNA
ladder).
554, SYG1519, and SYG1464 showed no gpa1 suppression activity in
either GU1 or GU2 cells. These constructs, then, did not display even
the very weak suppression seen with full-length SYG1, perhaps
due to altered protein conformation. The SYG1400 mutant,
in which all hydrophobic domains were eliminated, rescued gpa1 in both GU1 and GU2 as effectively as SYG1-1 (SYG1417). The SYG1-1 and SYG1400 rescued cells showed normal morphologies (Fig. 6, data not
shown for SYG1400). In contrast, deletion of 60 amino
acids from SYG1400 produced a surprising result. SYG1340 was capable of suppressing the growth arrest
associated with gpa1, but not the differentiation. These cells
were able to undergo mitosis while maintaining a shmoo morphology (Fig. 6). Deletion of another 141 amino acids (SYG1199) prevented any rescue.
417 mutant is marked with an
* to indicate that this is the SYG1-1 construct originally
isolated. For full-length SYG1, +w indicates
very weak suppression (see text). The dotted region must be
removed for full gpa1 suppression. The black region
is needed for suppression of differentiation (shmooing), and the diagonally striped region is needed for suppression of cell
cycle arrest.
340, which
are designated pSYG1-1 and pSYG1340, respectively. Cells which
lost the maintenance plasmid (pLGC) were selected and then grown on YPD
for 1 or 2 days. Photos were taken using a Nikon FXA microscope using a
60 objective and DIC (Nomarski) optics. Similar results were
obtained using strain GU2.
Subcellular Localization of Full-length and Truncated
Syg1p
Initially, we hypothesized that the dramatic increase in gpa1 suppressor activity of SYG1400 compared to
full-length SYG1 was attributable to altered subcellular
localization of the truncated protein. To test this model, crude
extracts containing epitope-tagged Syg1400p and Syg1p were
subjected to differential centrifugation. It should be noted that the
epitope fusion product of SYG1400 retained gpa1 suppression activity (data not shown). Immunological analysis of
marker proteins confirmed organellar separation. Surprisingly, both
Syg1400p and Syg1p localized to the particulate fraction, which
was greatly enriched for plasma membrane (Fig. 7A).
Therefore, altered subcellular localization of Syg1400p does not
account for its potent gpa1 suppression.
400p and Syg1p. Crude cellular extracts obtained from SP1
cells transformed with pADCLX (lane 1), pADCLX-SYG1400 (lane 2), and pADCLX-SYG1 (lane 3) were subjected to
differential centrifugation. Approximately 35 µg of the 10,000
g pellet and supernatant were analyzed for expression
of the epitope-tagged gene products as well as the organellar marker
proteins (PM, plasma membrane,
Na-K
-ATPase; cytosol,
glucose-6-phosphate dehydrogenase; ER, endoplasmic reticulum,
Sec63p). Indicated to the left are the apparent sizes of the
molecular mass standards in kDa (Bio-Rad, low range, prestained). Syg1p
migrated consistent with its predicted molecular mass of 104 kDa.
Depicted here, Syg1
400p migrates slower than its predicted
molecular mass of 47 kDa. However, using Sigma prestained standards,
Syg1400p migrates consistent with its predicted molecular mass. B, solubilization of Syg1400p from the particulate 10,000
g fraction. The immunoblot shows that only 1% SDS was
capable of releasing Syg1400p from the particulate fraction,
whereas 0.5 M NaCl, 0.5% Triton X-100, 2 M urea, 10
mM Tris-HCl, pH 10.5, and 0.5% -mercaptoethanol did not.
The Triton X-100 insolubility was also observed using 2% Triton X-100
(data not shown). After treatment with the solubilizing reagant, the
samples were centrifuged at 10,000 g for 10 min to
yield pellet (P) and supernatant (S)
fractions.
400p may associate with
another membrane-localized protein or contain its own membrane
targeting signal, even though no such signal is predicted by the
sequence. Treatment of the plasma membrane-enriched fraction with
reagents (high salt, high pH, urea and -mercaptoethanol) that
typically disrupt peripheral protein-protein interactions did not
release Syg1400p (Fig. 7B). Triton X-100 and SDS
were used to detect the presence of protein-membrane interactions. 1%
SDS completely solubilized Syg1400p, but 2% Triton X-100 was
unable to do so for both Syg1400p (Fig. 7B) and
Syg1p (data not shown).SYG1-1 Is a High Copy Suppressor in Mutant and Wild Type
Cells
The ability to rescue gpa1 requires high level
expression of SYG1-1. This was demonstrated by expressing SYG1-1 on a centromere-based plasmid that is maintained as a
low copy episome. This construct did not suppress gpa1,
demonstrating that high levels of Syg1-1p are needed.2 had a strong inhibitory effect. Thus, for all of these signaling
components, we found no evidence that message regulation is
significantly altered by SYG1-1.
-factor was used for all halo assays except sst1 and sst2 cells, for which 0.2 µg was used (see
``Materials and Methods'' for
details).
-factor (30) while Sst2p functions in an
independent pathway(21, 31, 32) .
Overexpression of SYG1-1 in sst1 or sst2 cells gave turbid halos with diameters that were unchanged (Fig. 8, C and D). These data indicated that SYG1-1 promotion of pheromone recovery is independent of Sst1p
and Sst2p. receptorless strains (33) . Thus,
our data confirm that gpa1 cells are indeed mating competent.
SYG1-1 Suppresses Excess Wild Type STE4 but Not a
Dominant Active STE4
To genetically localize the effects of SYG1-1 action, we examined the ability of this allele to
suppress the effects of high levels of wild type STE4. The
overexpression of STE4 is known to give a phenotype similar to
that of the gpa1 mutation, and it can be suppressed by the
overexpression of GPA1(10) . We used an inducible
construct in which STE4 overexpresssion and the resulting cell
cycle arrest were triggered by galactose. As seen in Fig. 9, SYG1-1 was able to suppress the cell cycle arrest caused by STE4 overexpression, although the full-length SYG1 clone was not.
, a dominant mutant in the G
subunit(8) . The STE4 mutant is lethal
because the mutant protein is less able to bind Gpa1p and thereby
escapes negative regulation, resulting in a constitutive signal for
cell cycle arrest(9) . This mutation is suppressed by the
overexpression of GPA1(34) or by the expression of
the MAT
locus when an a strain is
used(8) . Strain DBC was used to test for suppression of STE4. Although both MAT
2 and MCM1 constructs allowed for plasmid exchange, neither SYG1-1 nor SYG1 overexpression did (Fig. 10),
indicating that STE4 is epistatic to SYG1-1 overexpression. The inability of SYG1-1 to block the
effects of STE4
is likely due to the activation
mutation itself (Gly to Asp at residue 124). This alteration appears to
affect directly the interaction of Ste4p with Gpa1p (9) and
might also prevent protein-protein interactions critical for SYG1-1-mediated suppression. The STE4
result, in conjunction with the overexpressed STE4 data,
suggested that Syg1-1p suppresses gpa1 by interacting with
Ste4p.
. DBC cells carrying the STE4
mutation were transformed with the
indicated plasmids (vector = pADNS, MCM1 = YEp-MCM1, SYG1-1 = pADNS-SYG1-1, SYG1 = pADNS-SYG1,
2 = YEp-2). They
were grown in liquid media for 2 days and plated on selection media
(SC-leucine-arginine+canavanine). Growth requires loss of the
pU2C plasmid.
Suppressing Forms of Syg1p Can Physically Interact with
Ste4p
The ability of Syg1-1p to bind Ste4p was examined using
the two-hybrid reporter system(35) . The LexA-coding
sequence was fused to the carboxyl terminus of Syg1400p, and the
resulting hybrid construct was shown to retain gpa1 suppression activity (amino-terminal fusion constructs lost this
function). Fig. 11shows that hybrid fusions of Syg1400p or
Gpa1p can interact with Ste4p to induce expression of a HIS3 reporter gene. The same constructs also induced lacZ expression as judged by a filter lift -galactosidase assay
(data not shown).
400p and
Ste4p using a HIS3 reporter. Four independent L40
transformants were patched onto plasmid-selecting media (SC-Leu-Trp) and then replica plated to the same media or to
media that also selected for His (SC-Leu-Trp-His). The latter was double replica plated to
eliminate background growth. Following are the plasmids that correspond
to the indicated nonfusion (NF) vectors and fusion gene
products. For the GAL4 activation column: pGAD2F.N
(Gal4[768-881] only), pKB40.1
(Gal4[768-881]-Ste4p), and pGAD424-STE18
(Gal4[768-881]-Ste18p). For the LexA DNA
binding column: pBTM116 (LexA only), pCLA-SYG1
400
(Syg1-1p-LexA), and pBTM116-GPA1
(LexA-Gpa1p).
400p and Ste4p
was quantitatively analyzed (Table 3). This combination gave rise
to signals that were consistently 10-fold above all combinations of
vectors and non-interacting controls. However, signals for Gpa1p with
Ste4p, which were similar to what has been reported(18) , were
significantly higher than those for Syg1400p with Ste4p. This
presumably results in part from Syg1400p-LexA, like Syg1400p,
having a strong propensity for membrane attachment and thereby being
resistant to nuclear localization. It may also reflect an inherently
lower binding affinity. Also, although Syg1400p does not appear to
interact with Ste18p (Fig. 11, Table 3), this or some
other protein may strengthen Ste4p/Syg1400p binding.
signaling, can be suppressed by overexpression
of MCM1, MAT
2, and SYG1-1. MCM1 and MAT2 are both known to encode transcription
factors that control the expression of haploid specific genes such as GPA1, STE4, and STE18. Their ability to
suppress STE4 is consistent with their mode of
action being at the level of altered gene expression. In the case of MAT
2 expression in a cells, gpa1 suppression has been described previously (36) and can be
explained by the action of Mata1p/Mat2p heterodimers which
are known to repress transcription of haploid-specific
genes(1) . For MAT2 expression in
cells and for MCM1 expression in a cells, however, the
basis for gpa1 suppression is not entirely clear. Both MCM1 and MAT2 have the capacity to work
as heterodimeric partners and can act through interactions with other
transcription regulating proteins(37) . Therefore, increased
amounts of these proteins may result in a stoichiometric imbalance
resulting in blocked expression of signaling components or induced
expression of factors that suppress the mating pathway.. The MSG5 gene encodes a protein phosphatase that, when
overexpressed, is apparently able to counteract some protein
phosphorylation events that propagate the mating signal(38) .
Expression of a mutant a-factor receptor (STE3
) in a cells also
suppresses the gpa1 mutation(39) . The gpa1 suppressing action of STE3
is
carried out downstream of G
and results in altered FUS1 expression (36) . Other, uncharacterized gpa1 suppressors have also been reported(38, 40) .
400 to suppress the gpa1 mutation and overexpressed STE4 is most easily
explained by their gene products' binding and sequestering Ste4p
(G). This interaction is at least partially dependent on residue
124 of Ste4p since the dominant STE4 mutant
allele is not suppressible. Interestingly, this residue is also
involved either directly or indirectly in Gpa1p
interactions(9) . Although full-length Syg1p probably has Ste4p
binding capability (high copy SYG1 is a weak suppressor of gpa1), the removal of putative transmembrane domains from
Syg1p is required for its strong suppression of the pheromone pathway.
400p localizes to the same plasma membrane-enriched
subcellular fraction as does Syg1p. That Syg1400p can not be
released by a panel of reagents that disrupt protein-protein
interactions suggested that it is tightly associated with the plasma
membrane. Furthermore, both Syg1400p and Syg1p were insoluble in
the nonionic detergent Triton X-100, which disrupts hydrophobic but
neither polar, protein-protein, nor protein-lipid
interactions(41) , thereby suggesting that the
NH-terminal portion of Syg1p is sufficient to confer this
characteristic upon Syg1p. The overall solubility profiles for
Syg1400p and Syg1p may be indicative of protein-protein
interactions involving the cytoskeleton or membrane
skeleton(42) . Any interacting partner would, of course, need
to be sufficiently abundant to accommodate the high levels of
overexpressed Syg1400p and Syg1p. Alternatively, the Triton X-100
insolubility of Syg1400p and Syg1p may be an inherent
characteristic, perhaps occurring by self-aggregation or by complexing
with other insoluble material. Further biochemical studies should
reveal the basis of Syg1400p association with the plasma membrane
and may help determine both the normal function of Syg1p and how
G relates to that function.
heterodimers bind tightly to G
proteins and
mediate receptor coupling. Mammalian G heterodimers are also
involved in numerous cellular processes mediated by direct interactions
with a variety of downstream effectors including some forms of adenylyl
cyclase(43) , ion channels(44, 45) , phosducin (46) , PI3 kinase(47) , and
-adrenergic receptor
kinase(48, 49) . As in mammalian cells, there may be
multiple effectors responding to multiple G heterodimers in
yeast. Data from studies of Gpa2p, a Gpa1-related G
protein known
to activate adenylyl cyclase in yeast (50) , support the
existence of other G complexes. GPA2 is expressed
not only in haploid cells but also in diploids which are devoid of
Ste4p and Ste18p. In addition, overexpression of GPA2 does not
suppress gpa1(51) . These findings suggest that
another G
may indeed exist in yeast, a premise which should
be settled by the S. cerevisiae genome sequencing project. We
are currently using synthetic lethal and yeast two-hybrid screens to
elucidate the normal function of Syg1p, to identify functionally
redundant gene products, and to test whether interactions with Ste4p,
or any other G
or G proteins, play a role in Syg1p
function.
do not significantly disrupt signaling by RAS2 in wild type yeast cells (55, 56) and,
yet, have proven directly relavent to normal Ras
signaling(57) .
340 to
suppress the growth arrest phenotype of gpa1 cells, but leave
them in a morphologically altered state resembling that induced by
pheromone. These cells exhibited the general features associated with
shmoos, including enlargement and elongation, but they were budded and
continued to undergo mitosis. They showed some of the morphological
characteristics seen in fus3 or far1 cells that have
been treated with pheromone(58, 59) . This phenotype
may have resulted because the truncated protein binds Ste4p less
efficiently and therefore suppresses less well, falling below the
threshold for blocking differentiation. It is also possible that
Syg1340p has the capacity to promote cell division in an otherwise
differentiated cell. These SYG1-1 and SYG1340 suppressed gpa1 cells should be useful tools for studying
the components of the differentiation pathway and G protein signaling
and may relate to differentiation processes in higher eucaryotes. or G. Indeed, our observations are of particular
note because the identity of possible G
effectors has
remained elusive and because this is the first demonstration of an
interaction between Ste4p (G
) and a yeast protein which is not a G
protein subunit. The tight localization of Syg1p to a membrane fraction
involves additional features beyond the eight identified transmembrane
sequences, and this localization is consistent with a role in G protein
interactions. In addition, the identification of genes structurally
related to SYG1 suggests the existence of a family of genes
with shared functional characteristics. Finally, truncation mutants of SYG1 should continue to prove useful in the analysis of
signals leading to cell cycle arrest and differentiation and may also
help reveal other G protein-mediated pathways.
))
We thank George Sprague, Steven Reed, Duane Jenness,
Jeremy Thorner, David Meyer, Doreen Ma, Henrik Dohlman, and Malcolm
Whiteway for providing various reagents and for useful discussions. We
also thank Susanne Flückiger Staub for technical
assistance and Greg Payne and Harvey Herschman for their critical
reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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D. Hamburger, E. Rezzonico, J. MacDonald-Comber Petetot, C. Somerville, and Y. Poirier Identification and Characterization of the Arabidopsis PHO1 Gene Involved in Phosphate Loading to the Xylem PLANT CELL, April 1, 2002; 14(4): 889 - 902. [Abstract] [Full Text] [PDF]
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 F. K. Yoshimura, T. Wang, and S. Nanua Mink Cell Focus-Forming Murine Leukemia Virus Killing of Mink Cells Involves Apoptosis and Superinfection J. Virol., July 1, 2001; 75(13): 6007 - 6015. [Abstract] [Full Text] [PDF]
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 N. Ogawa, J. DeRisi, and P. O. Brown New Components of a System for Phosphate Accumulation and Polyphosphate Metabolism in Saccharomyces cerevisiae Revealed by Genomic Expression Analysis Mol. Biol. Cell, December 1, 2000; 11(12): 4309 - 4321. [Abstract] [Full Text]
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F. K. Yoshimura, T. Wang, F. Yu, H.-R. C. Kim, and J. R. Turner Mink Cell Focus-Forming Murine Leukemia Virus Infection Induces Apoptosis of Thymic Lymphocytes J. Virol., September 1, 2000; 74(17): 8119 - 8126. [Abstract] [Full Text]
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M. Marin, C. S. Tailor, A. Nouri, S. L. Kozak, and D. Kabat Polymorphisms of the Cell Surface Receptor Control Mouse Susceptibilities to Xenotropic and Polytropic Leukemia Viruses J. Virol., November 1, 1999; 73(11): 9362 - 9368. [Abstract] [Full Text] [PDF]
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 J.-L. Battini, J. E. J. Rasko, and A. D. Miller A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: Possible role in G protein-coupled signal transduction PNAS, February 16, 1999; 96(4): 1385 - 1390. [Abstract] [Full Text] [PDF]
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J. A. Levy Xenotropism: The elusive viral receptor finally uncovered PNAS, February 2, 1999; 96(3): 802 - 804. [Full Text] [PDF]
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C. S. Tailor, A. Nouri, C. G. Lee, C. Kozak, and D. Kabat Cloning and characterization of a cell surface receptor for xenotropic and polytropic murine leukemia viruses PNAS, February 2, 1999; 96(3): 927 - 932. [Abstract] [Full Text] [PDF]
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 S. J. Dowell, A. L. Bishop, S. L. Dyos, A. J. Brown, and M. S. Whiteway Mapping of a Yeast G Protein ß{gamma} Signaling Interaction Genetics, December 1, 1998; 150(4): 1407 - 1417. [Abstract] [Full Text]
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E. Li, E. Meldrum, H. F. Stratton, and D. E. Stone Substitutions in the Pheromone-Responsive Gss Protein of Saccharomyces cerevisiae Confer a Defect in Recovery from Pheromone Treatment Genetics, March 1, 1998; 148(3): 947 - 961. [Abstract] [Full Text] [PDF]
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T. Jonassen, M. Proft, F. Randez-Gil, J. R. Schultz, B. N. Marbois, K.-D. Entian, and C. F. Clarke Yeast Clk-1 Homologue (Coq7/Cat5) Is a Mitochondrial Protein in Coenzyme Q Synthesis J. Biol. Chem., February 6, 1998; 273(6): 3351 - 3357. [Abstract] [Full Text] [PDF]
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D.-Y. Jin, H. Teramoto, C.-Z. Giam, R. F. Chun, J. S. Gutkind, and K.-T. Jeang A Human Suppressor of c-Jun N-terminal Kinase 1 Activation by Tumor Necrosis Factor alpha J. Biol. Chem., October 10, 1997; 272(41): 25816 - 25823. [Abstract] [Full Text] [PDF]
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J. E. Hirschman, G. S. De Zutter, W. F. Simonds, and D. D. Jenness The Gbeta gamma Complex of the Yeast Pheromone Response Pathway. SUBCELLULAR FRACTIONATION AND PROTEIN-PROTEIN INTERACTIONS J. Biol. Chem., January 3, 1997; 272(1): 240 - 248. [Abstract] [Full Text] [PDF]
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T. Q. Do, A. Y. Hsu, T. Jonassen, P. T. Lee, and C. F. Clarke A Defect in Coenzyme Q Biosynthesis Is Responsible for the Respiratory Deficiency in Saccharomyces cerevisiae abc1 Mutants J. Biol. Chem., May 18, 2001; 276(21): 18161 - 18168. [Abstract] [Full Text] [PDF]
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