From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050
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ABSTRACT |
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Arf proteins are ubiquitous, eukaryotic regulators of virtually every step of vesicular membrane traffic. ADP-ribosylation factors are essential in yeast and the lethality resulting from either overexpression or underexpression (deletion) of Arf genes has previously been ascribed to dysregulation of the secretory process. We have identified a family of four genes (Suppressors of Arf ts, SAT) as high copy suppressors of a loss of function allele of ARF1 (arf1-3). Those proteins with SAT activity were found to contain a minimal consensus motif, including a C2C2H2 cluster with a novel and specific spacing. Genetic interactions between members of this family and with ARF1 are consistent with each sharing a common cellular pathway. Included in this family is Gcs1, a protein previously described (Poon, P. P., Wang, X., Rotman, M., Huber, I., Cukierman, E., Cassel, D., Singer, R. A., and Johnston, G. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10074-10077) to possess Arf GTPase-activating protein (GAP) activity, demonstrating a direct interaction between Arf and at least one of these suppressors. The suppression of the loss of Arf function by overexpression of Gcs1 and demonstration of direct, preferential binding of Gcs1 to the activated form of Arf (Arf·GTP) lead us to conclude that the biological role of Gcs1 is as an effector of the essential function of Arf in mitotic growth, rather than a down-regulator as implied by the biochemical (Arf GAP) activity.
Suppression of the growth defect of
arf13 cells was observed under
conditions that did not alter the secretory defect associated with
arf1
mutation, indicating that the
essential role of Arf in eukaryotes can be distinguished from role(s)
in the secretory pathway and appear to employ distinct pathways and
effectors.
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INTRODUCTION |
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ADP-ribosylation factors
(Arfs)1 are the family of
monomeric, 21-kDa GTP-binding proteins originally identified as protein co-factors for cholera toxin-catalyzed ADP-ribosylation of
Gs, the heterotrimeric G protein activator of adenylyl
cyclase (1, 2). Studies have implicated Arfs as regulators of a number of steps of vesicular membrane transport, coat protein assembly, and
maintenance of the integrity of the ER and Golgi compartments (3-7).
Studies documenting the direct activation of phospholipase D (PLD)
activity by Arfs and effects of PLD on an in vitro Golgi transport assay have led to the controversial conclusion that PLD(s)
mediate the action of Arfs on membrane traffic (8). An obligate role
for Arf-sensitive PLD is doubtful, however, as Arfs are essential in
yeast (Saccharomyces cerevisiae), Arf mutations affect the
same organelles in the secretory pathway in yeast that they do in
cultured mammalian cells, and yet there is no Arf sensitive PLD
activity in yeast (28). The ability of each of the five human Arfs to
complement the lethal, double ARF deletion
(arf1arf2
) in yeast
highlights the evolutionary conservation of function(s) of Arfs and has
prompted the use of genetic studies to define the regulatory pathway
controlled by Arf proteins, specifically the essential role of Arfs in
eukaryotic cell growth.
Either overexpression or deletion of (both) ARF genes is
lethal to yeast cells (9). The two yeast Arf proteins are 96% identical, and no phenotype has been defined for the loss of
ARF2. In contrast, arf1 cells grow
slower than parental controls at all temperatures are weakly
cold-sensitive (cs), defective in the ability to process
secreted proteins, e.g. invertase, and are supersensitive to
fluoride (9, 10). Supersensitivity to fluoride results from an unknown
mechanism, but it has proven a useful and specific indicator of loss of
Arf1 function. A second copy of ARF2 complements these
phenotypes associated with arf1
cells. The
5-10-fold higher level of expression of Arf1 over Arf2 is the
likely explanation for the differences in phenotypes between
arf1
and arf2
(11). For this reason, we focused our efforts on genetic studies of
ARF1, and the studies described below were conducted in an arf2
background, unless otherwise
indicated.
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MATERIALS AND METHODS |
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Yeast Culture
Yeast were cultured using standard conditions, as described in Sherman et al. (12). Selective plates containing fluoride were prepared according to Stearns et al. (11). Yeast transformations were performed by the PEG/LiCl method of Schiestl et al. (13).
Mutagenesis
PCR Mutagenesis-- Random mutagenesis of the open reading frame of the ARF1 gene was achieved by PCR under conditions of reduced stringency; including 50 µM dATP and 0.1 mM MnCl2, according to Leung et al. (14). Plasmids bearing the mutated ARF1 were produced in yeast by co-transfecting the mutant PCR products and gapped plasmid, pJCY1-31 (10), to allow gap repair by homologous recombination. The strain used, RT166, was deleted for both ARF1 and ARF2 and carried human Arf4 on another plasmid, under control by the GAL1 promoter. Conditional alleles of ARF1 were selected after replica plating transformants onto YEPD (1% yeast extract, 2% bacto-peptone, 2% glucose) plates (to turn off expression of human Arf4) and growing cells at 16, 28, and 37 °C. The arf1-3 mutation was introduced at the ARF1 locus by homologous recombination with replacement of the arf1::HIS3 allele.
Site-directed Mutagenesis-- Specific mutations were introduced into plasmids using gene-specific primers encoding the desired changes with at least 18 bp of priming nucleotides in PCR reactions, as described in Kahn et al. (10). All PCR amplified fragments were completely sequenced both to confirm the introduction of desired changes and to ensure no additional changes were produced.
Gene Cloning and Deletion
A high copy, Arf-deleted, genomic library was constructed using
genomic DNA (15) from RT166
(arf1arf2
pGAL1-hArf4)
that was partially digested with Sau3A before size selection
(3-20 kilobase pairs) and ligation into the BamHI site of
YEp352, a high copy plasmid bearing the URA3-selectable
marker.
The six yeast genes tested for SAT activity were amplified from genomic DNA by PCR using gene specific primers and cloned into the high copy (2 µ-containing, URA3-marked) plasmid, YEp352. The lengths of 5'- and 3'-UTRs amplified varied with the distance to the adjacent genes. The length of 5' upstream, open reading frame and 3' downstream regions amplified were: SAT1 = 427/1449/404 bp; SAT2 = 220/896/158 bp; GCS1 = 243/1058/475 bp; GLO3 = 320/1481/128 bp; SPS18 = 162/902/201 bp; and GTS1 = 301/1190/880 bp.
The sat1::HIS3 and sat2::HIS3 deletions were generated by the method of Baudin et al. (16) after PCR amplification of the HIS3 gene using primers that encode 40 bp of gene-specific sequence from each end of the open reading frames. Auxotrophic markers were then "swapped" as described in Cross (27) to allow the ready isolation of double and triple deletants. Each deletion allele and swapped marker was confirmed by PCR.
Invertase Assay
The assay for processing of invertase was performed as described in Stearns et al. (9). Cells were grown in medium containing a high concentration (5%) glucose to log phase before being collected by centrifugation, washed, and resuspended into YEPD containing 0.1% dextrose. Half of the culture was then incubated at 30 °C and the other half at 37 °C. After 3 h, cells (2 A600 units) were collected, rinsed in 25 mM Tris-Cl, pH 7.4, 10 mM NaN3, 1 mM phenylmethylsulfonyl fluoride, and lysed by agitation with glass beads in Laemmli's sample buffer before boiling for 5 min. Proteins were fractionated on 7.5% polyacrylamide-SDS gels before transfer to nitrocellulose membranes. The primary antibody used was a guinea pig anti-invertase polyclonal antiserum (the generous gift of D. Preuss), and the secondary antibody was goat anti-guinea pig IgG coupled to peroxidase. Detection of signal was accomplished with the enhanced chemiluminescence kit (ECL) from Amersham Pharmacia Biotech.
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RESULTS AND DISCUSSION |
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The arf1-3 Allele Is ts--
A temperature sensitive
(ts) allele of ARF1, termed arf1-3,
was generated by chemical mutagenesis of plasmids bearing the
ARF1 gene and selection for sensitivity to growth at
37 °C. arf1-3 is a conditional loss of function mutation
that is recessive to both ARF1 and ARF2.
Sequencing of the open reading frame of arf1-3 revealed a
single base pair mutation resulting in a mis-sense mutation,
[T32I]Arf1. This residue is just downstream of the first consensus
GTP binding domain, G24LDGAGK30, and
immediately adjacent to Thr31, whose mutation results in a
negative dominant phenotype (17). Thr32 lies in the
nucleotide binding pocket of Arf1·GDP (18), 2.8 Å from an
-phosphate oxygen such that the introduction of the larger side
chain of an isoleucine is predicted to disturb the nucleotide binding.
Quantitative immunoblotting revealed only minor differences in the
level of Arf1-3 versus Arf1, which were not affected by
growth at the restrictive temperature. Thus, altered stability of the
mutant protein does not explain the phenotype.
Cloning a Suppressor of arf1-3-- A yeast genomic 2 µ library was screened for high-copy suppressors of arf1-3 at 37 °C. From 300,000 colonies screened, we obtained nine temperature-resistant (tr) colonies, all of which grew at near wild type rates but reverted to ts with loss of the library plasmid. Sequencing the inserts and comparison with the Saccharomyces genome data base (SGD) revealed the presence of a single, complete open reading frame present in all nine plasmids, labeled YDR524C in the SGD, and named suppressor of Arf1 ts or SAT1.2 Suppressor activity of SAT1 was confirmed with a high copy (2 µ) plasmid bearing the entire gene, obtained by PCR amplification of a 2.3-kilobase pairs genomic fragment, including the open reading frame of 1446 and 404 bp and 427 bp upstream and downstream, respectively (see Fig. 1). The same genomic fragment failed to complement arf1-3 when present on a low copy (CEN) plasmid.
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Sat1 Contains a Cysteine Repeat (Zinc Finger) Motif--
SAT1
encodes a protein 482 residues in length, with a predicted molecular
mass of 54 kDa. The DNA sequence of the open reading frame of one of
our genomic library inserts was identical to the SGD entry for YDR524C.
BLAST analyses of Sat1 against the SGD revealed weak homology between
Sat1 and a group of yeast proteins with a cysteine-rich zinc finger
motif, one of which (GCS1) has been reported to possess Arf
GTPase-activating protein (GAP) activity (19). The homologies between
Sat1 and the members of this group, including Gcs1, Glo3, YIL044C,
Gts1, and Sps18, are limited to the N-terminal, cysteine-rich, zinc
finger domain, abbreviated as C2C2 (see Fig.
3). This is the region identified by Ireland et al. (20) in
Gcs1, Glo3, and Sps18 and implicated in Arf GAP activity (19). The
cysteine-rich domain of Sat1 is removed from the N terminus, by about
160 residues, with respect to the others. A similar degree of homology,
again limited to the zinc finger domain, was observed in a number of
proteins from other species, including -centaurin and a mammalian
Arf GAP, described by Cassel and colleagues (19, 21). We next explored
the possibility that other members of the group of yeast proteins with
the cysteine-rich motif possess effector activity for Arf1.
The C2C2H2 Motif Is a Minimal Domain Required for Sat Activity-- To test the importance of cysteine and histidine residues to SAT activity, mutants of the first and second cysteines and the two histidines were constructed in Sat1 and tested for suppressor activity. Mutation of the second cysteine in the motif in gcs1 (termed gcs1-1) to tyrosine results in a cs failure to re-enter the cell cycle phenotype, also observed with deletion of GCS1 (20, 22). As seen in Fig. 2, the [C186A]Sat1 or [C189Y]Sat1 mutants have lost SAT activity. Similarly, mutation of both of the histidines to residues present in Sps18 ([H214L, H220N]Sat1) also caused the loss of SAT activity. Thus, we propose a minimal consensus sequence for SAT activity to include the histidines, yielding the motif: C-X2-CX16-17-C-X2-C-X3-H-X5-H. No other proteins containing this exact motif were found in either the yeast or Swiss-Prot data bases, although 22 others were found in SGD to contain the four cysteines with this spacing. Multiple human expressed sequence tags (ESTs) from distinct genes were found to contain the Sat consensus (C2C2H2) and include more extensive homology to Sat1 in the surrounding region than is present in the yeast family members. These human proteins are currently being tested for SAT activity.
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Genetic Interactions between the SAT Proteins and with arf1--
A
null allele of SAT1 was constructed in both haploid and
diploid cells by replacement of the open reading frame of
SAT1 with the HIS3 gene, as described under
"Materials and Methods." SAT1 is not an essential gene,
as haploid strains of each mating type, carrying the disrupted gene,
were viable. This was confirmed by dissection of spores, in which
HIS3+ (sat1) segregated
2:2 and all four spores were viable. Haploid strains deleted for
SAT1 were found to grow at the same rates as wild type
strains at 16, 28, or 37 °C and grew well on nonfermentable carbon
sources, e.g. glycerol (not shown). Diploid cells homozygous for the deletion also appeared normal and sporulated as well as controls.
Separation of Secretory and Mitotic Growth Defects--
The
sat1 deletion alone had no obvious effect on
the secretory pathway, as visualized by the processing of invertase
during its transit of the secretory pathway, at either 30 or 37 °C
(Fig. 4; WT versus sat1). In
contrast, the deletion of ARF1 has previously been shown to
cause a partial defect in invertase glycosylation, consistent with a
defect in transit through the Golgi (Ref. 9; Fig. 4, arf1
versus WT). Cells carrying the arf1-3 allele
processed invertase to the same (incomplete) level at permissive and
restrictive temperatures, comparable with that of
arf1
cells (see Fig. 4; compare arf1 to
arf1-3). This defect was unaffected by the introduction of
SAT1 on high copy plasmids at either temperature (arf1-3 ± SAT1). Thus, under restrictive conditions (37 °C)
that result in little or no cell growth (arf1-3) or near WT growth rates (arf1-3 + SAT1), there is no discernible difference in the ability to process invertase through the Golgi. The sec18-1 strain is
shown as a control that shows the ER form (core glycosylated) of
invertase as SEC18 encodes the yeast homolog of the NSF
protein, required for exit from the ER. Although we cannot completely
exclude the possibility that it is Arf acting at other membrane traffic sites (e.g. endocytosis) that is required for cell growth,
we consider this unlikely because the phenotypes observed previously with depletion of Arfs were most severe on the early secretory pathway
and readily observed with this same invertase assay (9). Thus, we
interpret these data as indicating an essential role for Arf proteins
in cell growth that can be separated from its role in membrane
transport and that Sat1 and related proteins are effectors of this
essential Arf function.
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ACKNOWLEDGEMENTS |
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We thank many of the members of the yeast community in the National Institutes of Health intramural program for helpful discussions during the course of this work. We also gratefully acknowledge the sharing of unpublished observations and discussions with Dr. Anne Thiebert and critical review of the manuscript by Drs. Annette Boman, Anita Corbett, and J. David Lambeth. The anti-invertase antibodies were the generous gift of Drs. Daphne Preuss and David Botstein.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM55823 and was begun in the intramural program of the NCI.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Laboratory of Molecular Embryology, NICHD, NIH,
Bethesda, MD 20892-5431.
§ To whom all correspondence should be addressed. Tel.: 404-727-3561; Fax: 404-727-3746; E-mail: rkahn{at}bimcore.emory.edu.
1 The abbreviations used are: Arf(s), ADP-ribosylation factor(s); ER, endoplasmic reticulum; PLD, phospholipase D; PCR, polymerase chain reaction; bp, base pair(s); SGD, Saccharomyces genome data base; GAP, GTPase-activating protein; WT, wild type.
2 As this appears to be the first functional description for this gene we propose the name SAT1 for YDR524C and SAT2 for YIL044C.
3 A. Thiebert, unpublished observations.
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REFERENCES |
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