Regulation of Phosphatidylglycerophosphate Synthase Levels in Saccharomyces cerevisiae*

Haifa ShenDagger and William Dowhan§

From the Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77225

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
References

The PGS1 gene of Saccharomyces cerevisiae encodes phosphatidylglycerophosphate (PG-P) synthase. PG-P synthase activity is regulated by factors affecting mitochondrial development and through cross-pathway control by inositol. The molecular mechanism of this regulation was examined by using a reporter gene under control of the PGS1 gene promoter (PPGS1-lacZ). Gene expression subject to carbon source regulation was monitored both at steady-state level and during the switch between different carbon sources. Cells grown in a non-fermentable carbon source had beta -galactosidase levels 3-fold higher than those grown in glucose. A shift from glucose to lactate rapidly raised the level of gene expression, whereas a shift back to glucose had the opposite effect. In either a pgs1 null mutant or a rho mutant grown in glucose, PPGS1-lacZ expression was 30-50% of the level in wild type cells. Addition of inositol to the growth medium resulted in a 2-3-fold reduction in gene expression in wild type cells. In ino2 and ino4 mutants, gene expression was greatly reduced and was not subject to inositol regulation consistent with inositol repression being dependent on the INO2 and INO4 regulatory genes. PPGS1-lacZ expression was elevated in a cds1 null mutant in the presence or absence of inositol, indicating that the capacity to synthesize CDP-diacylglycerol affects gene expression. Lack of cardiolipin synthesis (cls1 null mutant) had no effect on reporter gene expression.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
References

Biosynthesis of phosphatidylglycerol (PG)1 and cardiolipin (CL) from the precursor phosphatidic acid involves four sequential steps catalyzed by the enzymes CDP-diacylglycerol (CDP-DAG) synthase, phosphatidylglycerophosphate (PG-P) synthase, PG-P phosphatase, and CL synthase. The committed and rate-limiting step is catalyzed by PG-P synthase (1, 2), which is encoded by the PGS1 gene in Saccharomyces cerevisiae (3). PG and CL are primarily synthesized in the mitochondrial inner membrane and are limited in their distribution to the inner and outer membranes of the mitochondria in yeast (4-6). CL has been postulated to be important for the functioning of several mitochondrial enzymes and the import of proteins into the mitochondria (7-11). PG appears to substitute for potentially critical functions of CL, inasmuch as a 5-fold increase of PG levels can compensate for the loss of CL in mutants lacking CL synthase (cls1 null strains) (12-14), whereas absence of both PG and CL in mutants lacking PG-P synthase results in severe mitochondrial dysfunction (3, 15).

Previous genetic and biochemical studies have indicated that two sets of factors affect PG-P synthase activity: cross-pathway control by inositol and choline and factors affecting mitochondrial development such as carbon source, oxygen, and mutations in mitochondrial DNA (16, 17). Regulation by inositol is unique compared with its regulation of other phospholipid biosynthetic pathways in that a decrease in PG-P synthase activity is sudden and dramatic upon addition of inositol to the growth medium (16). It was speculated that this rapid effect could not be due to repression of gene transcription but, instead, is a result of inactivation and/or degradation of the PG-P synthase. In addition, PG-P synthase activity was found not subject to regulation by the INO2-INO4-OPI1 regulatory genes which are required for cross pathway regulation by inositol (16). The PGS1 gene (also known as the PEL1 gene) encoding PG-P synthase in yeast has been isolated and characterized (3, 18). Sequence analysis revealed that there is at least one putative UASINO element (upstream activating sequence responsive to inositol) 284 base pairs 5' to the putative PGS1 gene start codon, indicating that a common regulatory mechanism responsive to inositol may play a role in PGS1 gene expression (3).

Expression of mitochondrial-encoded genes and mitochondrial biosynthesis is mediated by cis-acting elements and regulatory proteins that respond to the carbon source supporting growth (19). Transcription levels of genes involved are rapidly adapted to changes in the available carbon source. Previous studies have shown there is correlation between mitochondrial development and cellular CL levels (4, 17, 20). Yeast cells growing in a non-fermentable carbon source have higher PG-P synthase activity and a higher CL content than cells grown in glucose. Cells growing in a medium with glucose as the carbon source have two phases of growth. During the fermentative growth phase when ethanol is accumulated at the expense of glucose, CL is maintained at a lower level. After glucose is completely consumed, yeast cells begin to use ethanol as a carbon source, and CL levels almost double within a short period of time (4). Carbon source mainly affects PG-P synthase activity, but not other enzymes in CL biosynthesis, such as PG-P phosphatase or CL synthase (17, 21, 22). The amount of PG-P synthase activity relative to mitochondrial protein is kept at the same level regardless of changes in growth conditions; thus, PG-P synthase activity is regulated coordinately with the amount of mitochondria per cell (2). However, the DNA sequence 5' to the PGS1 gene does not contain any consensus binding sites for regulatory factors involved in carbon source-dependent transcriptional regulation (19).

In this study, we focused on the molecular basis for the regulation of PGS1 expression by factors affecting mitochondrial development, by inositol, and by mutations in the other structural genes necessary for PG and CL synthesis. We demonstrate that PGS1 gene expression is regulated by factors affecting mitochondrial function, by inositol, and by the capacity of cells to synthesize CDP-DAG.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
References

Materials-- All chemicals were reagent grade or better. o-Nitrophenyl beta -D-galactopyranoside was from Sigma. Restriction endonucleases were from New England Biolabs. Polymerase chain reaction SuperMix was a product of Life Technologies, Inc. Oligonucleotides were prepared commercially by Genosys Biotechnologies. YEP broth and synthetic media for yeast growth and selection were from Bio 101, Inc. Yeast nitrogen base without amino acids was from Difco. The BCA kit was from Pierce.

Strains, Media, and Growth Conditions-- Yeast strains used in this study are listed in Table I. Yeast cultures were grown at 30 °C in synthetic minimal media (18) containing either 2% glucose, 2% galactose, 2% sodium lactate, or 3% glycerol with 0.95% ethanol as the carbon source. Where indicated, 10 µM or 70 µM inositol was added to growth medium.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Yeast strains and plasmids

Plasmid Construction-- Plasmid pSD70 contains the putative promoter of the yeast PGS1 gene fused to a lacZ reporter gene. It was created by replacing the EcoRI fragment of plasmid pMA109 (26) with the polymerase chain reaction-amplified region 5' to the PGS1 gene, which includes the putative UASINO element (3). Amplification of this region from yeast chromosomal DNA employed primer 1 (5'-GAAAGGAATTCTAGGTGATATTGC-3') and primer 2 (5'-GAGAATTCTGGAGCAAACGAGTCGTCAT-3'). They were designed according to the DNA sequence in the vicinity of the PGS1 gene (3). Primer 1 begins 331 base pairs 5' to the PGS1 start codon, and primer 2 ends at the 20th base pair in the PGS1 open reading frame. The final fusion plasmid includes a DNA fragment encoding the first 7 amino acids encoded by the PGS1 gene fused in frame with the lacZ gene. Plasmid pSD70 was introduced into yeast cells by transformation of CaCl2-treated cells (25).

Isolation of rho- Mutants-- Isolation of ethidium bromide-induced rho mutants was performed as described previously (28). Yeast cells were plated on a synthetic medium containing 10 µg/ml ethidium bromide and 2% glucose and grown at 30 °C for 48 h. Colonies were screened for by their inability to grow on a synthetic medium with 3% glycerol as the carbon source. Although these cells were assumed to be rho mutants, the only criterion used for their selection was their inability to grow on a non-fermentable carbon source.

Preparation of Cell Extracts and beta -Galactosidase Assay-- Preparation of cell extracts was modified from a previous report (25). All procedures were carried out at 4 °C. Yeast cells were harvested by centrifugation, washed in 100 mM sodium phosphate, pH 7.0, and resuspended in the same buffer. The cell suspension was mixed with an equal volume of prechilled glass beads (diameter 0.3 mm) and disrupted in a Mini-BeadbeaterTM (Biospec Products) by four 1-min bursts at 2,800 rpm with a 2-min pause between bursts. Glass beads and unbroken cells were removed by centrifugation at 1,500 × g for 10 min. beta -Galactosidase assays were modified from a previously described procedure (29). Briefly, 100 µl of cell extract was mixed with 700 µl of 100 mM sodium phosphate and 200 µl of 2 mg/ml o-nitrophenyl beta -D-galactopyranoside, and the mixture was incubated at 30 °C for 30 min to 2 h. The reaction was stopped by adding 1 ml of 1 M Na2CO3, and absorbance was measured at 420 nm. beta -Galactosidase activities reported are equal to 380 × the optical density at 420 nm produced/min/mg of total protein in cell extracts. Protein concentration in each cell extract was determined using a BCA protein assay kit with bovine serum albumin as the standard.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
References

Regulation of PGS1 Gene Expression by Carbon Source-- Carbon source affects CL content as a result of regulation of PG-P synthase activity and mitochondrial development. Because PG-P synthase catalyzes the rate-limiting step in CL biosynthesis, we examined whether carbon source regulation was mediated via gene expression by monitoring beta -galactosidase activity expressed from the PPGS1-lacZ reporter gene. Because mitochondrial development is more affected in the stationary phase of growth than in the exponential phase (30), and cellular PG-P synthase activity varies depending on growth phase (17), we used mid-log phase cells in this study.

PPGS1-lacZ expression was repressed in glucose medium, but derepressed in media with a non-fermentable carbon source (lactate or glycerol-ethanol) (Fig. 1). Strain DL1 (shown in Fig. 1) had 10% higher beta -galactosidase activity than strain YPH102 (not shown) when grown in glycerol-ethanol medium and 20% lower beta -galactosidase activity than strain YPH102 when grown in lactate medium; however, the overall dependence on carbon source was the same for both wild type strains. The 3-fold increase of PPGS1-lacZ expression was in line with PG-P synthase functional assay data, which showed that cells grown in glycerol-ethanol had 3-fold higher activity than cells grown in glucose (17).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1.   PPGS1-lacZ gene expression in response to carbon source and mitochondrial function. The parent strain DL1 (black box), pgs1 mutant YCD4 (open box), and a rho mutant derived from strain DL1 (gray box) all bearing plasmid pSD70 were grown to mid-log phase in an inositol-free medium with 2% glucose. Cell growth was either continued in or shifted to the same medium with either 2% lactate or 3% glycerol plus 0.95% ethanol as the carbon source. Cells were harvested after 4 h, and beta -galactosidase activity was determined in whole cell extracts. Each value represents the mean of three experiments.

We next studied PPGS1-lacZ expression during adaptation to changes in carbon source. Because yeast cells growing in minimal media tend to reach stationary phase before all the glucose is consumed, we facilitated the switch of a mid-log culture from fermentative growth to oxidative phosphorylation-dependent growth by shifting cells from glucose to lactate medium at time zero (Fig. 2, dashed lines). Cell growth monitored by cell density and increase in total cellular protein paused in the first hour after the shift; however, increase in beta -galactosidase activity was observed immediately after the switch. The highest activity was reached after 5 h of growth, which was 3-fold as high as in glucose-grown cells, indicating gene expression was fully derepressed at this stage. These results also match with the result in Fig. 1, showing cells grown in a non-fermentable carbon source were 3-fold higher in beta -galactosidase activity when compared with cells grown on glucose. Because cell density and total cellular protein only increased 10% within the first 5 h of growth, increase in the rate of gene expression probably exceeded that of mitochondria synthesis and overall cell growth.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Adaptation to non-fermentative and fermentative growth. Mid-log phase cells of strain DL1/pSD70 growing in an inositol-free medium with either 2% glucose (dashed lines) or 2% lactate (solid lines) were collected, washed with an inositol-free medium without carbon source, and resuspended at time zero in the same medium with either 2% lactate (dashed lines) or 2% glucose (solid lines), respectively; in the latter case, the glucose medium was supplemented either with (closed symbols) or without (open symbols) 10 mM cycloheximide. Cells were sampled at the indicated time points, cell density (A) was determined, and beta -galactosidase activity (B) was determined in whole cell extracts. Values are representative of three experiments.

Adaptation to catabolite repression was also followed by shifting a cell culture from lactate- to glucose-containing medium (Fig. 2, solid lines). We observed a dramatic decrease in beta -galactosidase activity once cells were in glucose medium with a steady-state level reached after 4 h of growth, which was 30-40% of the derepressed level. This result was surprising, because neither cell density nor total cellular protein increased significantly during the first 2 h of growth, while the drop in beta -galactosidase activity was significant. Treatment of cells with 10 mM cycloheximide during the shift of carbon source blocked cell growth and changes in beta -galactosidase activity in the cell extracts regardless of the presence of glucose in growth medium, indicating that beta -galactosidase is a stable enzyme in yeast cells when protein synthesis and growth are blocked. Because the chimeric protein has no structural or functional similarity with the PGS1 gene product, this rapid turnover must be related to general protein turnover occurring during adaptive growth. Foreign proteins may be even more susceptible than native proteins to such turnover.

Previous studies on carbon source regulation of many genes encoding proteins imported into the mitochondria have revealed that regulation is at the transcriptional level and involves a complex of transcription factors (31-33). The above results indicate that PGS1 gene expression is repressed in a fermentable carbon source and derepressed by a non-fermentable carbon source and that PG-P synthase activity levels respond to carbon source at least in part through regulation of gene expression. However, we do not know whether changes in mitochondrial biogenesis and function trigger gene expression or whether these affects are directly brought about by responses to the carbon source.

Effect of Mitochondrial Function on PGS1 Expression-- The pgs1 null mutant strain (YCD4) has severe defects in mitochondrial function (3, 18). In addition to its inability to utilize a non-fermentable carbon source for growth, this mutant also has a petite lethal phenotype (18), characterized by incompatibility with superimposition of extensive lesions in mitochondrial DNA such as in rho mutants. This strain along with a rho mutant served as experimental tools to study the impact of dysfunctional mitochondria on gene expression. The PPGS1-lacZ reporter gene was introduced into strain YCD4, and cells originally grown in glucose-containing medium were suspended in media with different carbon sources for the time indicated (Fig. 1). PPGS1-lacZ reporter gene expression in this mutant was only 30-40% of the wild type level when cells were in glucose-containing media. This activity was even lower when glucose was replaced with lactate or glycerol-ethanol (although no cell growth occurred in these latter carbon sources), suggesting net turnover of existing gene product in the absence of no new synthesis. The results were strikingly similar with that of gene expression in the rho mutant, which also cannot grow on non-fermentable carbon sources. The above result argues that functional mitochondria, and not simply carbon source, are a prerequisite for normal levels of PGS1 gene expression, which may be coordinately regulated with the expression of other mitochondrial proteins.

Regulation of PGS1 Expression by Inositol-- Inositol regulates the expression of enzymes in the major phospholipid biosynthetic pathways and has been shown to repress the level of PG-P synthase activity (1, 2); previous results (16) indicated that PG-P synthase may be regulated by inositol through a mechanism not related to the established modes of inositol regulation. However, sequence examination of the upstream region of the PGS1 gene does show the presence of an UASINO element that could serve as a cis-acting element in inositol-dependent regulation mediated by the INO2-INO4-OPI1 gene circuit. As shown in Fig. 3, PPGS1-lacZ gene expression was derepressed in inositol-free medium and was repressed by the presence of inositol. The degree of repression was not in parallel with inositol concentration, but was in agreement with the PG-P synthase functional assay results measured at steady state (16). The degree of repression was strain dependent with strain YPH102 (data not shown) being more sensitive to repression by 10 µM inositol than strain DL1.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3.   PPGS1-lacZ expression in ino2 and ino4 mutant strains. Wild type strain DL1 (black box), the ino2 mutant SH303 (gray box), and the ino4 mutant SH307 (open box) all bearing plasmid pSD70 were grown to mid-log phase in a minimal medium supplemented with 2% glucose and inositol as indicated. beta -Galactosidase activity was determined in whole cell extracts. Each value represents the mean of three experiments.

To determine the possible involvement of the INO2 and INO4 gene products, we introduced the reporter gene into ino2 and ino4 mutant strains. Because these mutant strains do not grow in inositol-free medium, 10 µM and 70 µM inositol was added to the growth media to support cell growth. Gene expression did not response to inositol regulation in the mutant strains, and beta -galactosidase activity in the mutants was close to the fully repressed level of wild type strain DL1 at 70 µM; strain DL1 grown in the absence of inositol had beta -galactosidase activity of 7.5 ± 1.0. Because gene expression in the ino2 and ino4 mutants was significantly reduced, our results indicate that INO2 and INO4 regulatory genes are also involved in PGS1 gene expression. This result appears to be in conflict with earlier reports of a too rapid reduction of PG-P synthase activity in response to inositol to be explained by regulation at the transcriptional or translational level (16). However, both results would be compatible with each other if regulation of PG-P synthase activity were subject to both transcriptional regulation (long term adaptation) and posttranscriptional regulation (short term response) such as modification or degradation. There is precedence for such a dual response to a common regulator of phospholipid metabolism, inasmuch as the level of CDP-DAG synthase activity regulates some gene products at the level of transcription (phosphatidylserine (PS) synthase), whereas others (phosphatidylinositol (PI) synthase) appear to be regulated through posttranslational events (27).

Expression of the PGS1 Gene in Mutants Affected in CL Biosynthesis-- In addition to PG-P synthase, CDP-DAG synthase (encoded by the cds1 gene) and CL synthase are also directly involved in CL biosynthesis in yeast (1, 2). Alteration of cellular CDP-DAG synthase activity in cds1 mutants affects several major phospholipid biosynthetic enzymes including PS synthase and PI synthase, which catalyze two immediate downstream reactions requiring CDP-DAG (27). Reduction in CDP-DAG synthase activity raises cellular PS synthase levels and causes constitutive expression of inositol-1-P synthase as a result of derepression of CHO1/PSS and INO1 gene expression, respectively (34, 35); this regulation is independent of the INO2 and OPI1 regulatory genes and therefore represents a novel mode of regulation of phospholipid biosynthetic genes independent of inositol (27). Reduction in PI synthase activity in response to low levels of CDP-DAG synthase appears to occur by posttranslational processes (27).

Inasmuch as PG-P synthase catalyzes another immediate step downstream of CDP-DAG synthesis (1, 2), we examined whether low CDP-DAG synthase activity would also affect PGS1 gene expression. The PPGS1-lacZ reporter gene was introduced into the cds1 null mutant bearing a plasmid expressing a human cDNA encoding a CDP-DAG synthase (36), which is sufficient to sustain cell growth if induced from the PGAL1 promoter, but does not correct the other phenotypes associated with low levels of CDP-DAG synthesis (27). PPGS1-lacZ expression was higher in the cds1 null mutant than in the wild type strain, and the degree of repression in the presence of inositol was not as severe in the mutant strain as in the wild type (Fig. 4). This result mimics the results observed for CHO1/PSS gene expression in a cds1 point mutant strain and in cds1 null strains expressing the human cDNA under the above conditions (34, 35). Therefore, PGS1 gene expression also responds to an inositol-independent regulation dependent on the capacity of cells to synthesize CDP-DAG.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   PPGS1-lacZ gene expression in cds1 and cls1 mutant strains. The cds1 mutant YSD90A/phCDS transformed with plasmid pSD70 (gray box) was grown to mid-log phase in minimal medium with 2% galactose (to induce the plasmid borne copy of the CDS gene) and the indicated inositol level. beta -Galactosidase activity was assayed in whole cell extracts and compared with that of the wild type strain YPH102 (black box) grown in the same medium. The cls1 mutant (YCD2) with plasmid pSD70 was grown in a minimal medium with 2% glucose and the indicated inositol. beta -Galactosidase activity of the mutant (open box) was compared with that of the wild type strain YPH98 (black box) grown in the same medium. Each value represents the mean of three experiments. Activity of the wild type strains in inositol-free medium was set at 100%.

The cls1 null mutant grows on both fermentable and non-fermentable carbon sources although it does appear to be somewhat compromised in growth on the latter substrates (12-14). PPGS1-lacZ reporter gene was introduced into the cls1 mutant to test whether this less stringent effect of carbon source on growth (versus pgs1 or rho mutants) also affects PGS1 gene expression. beta -Galactosidase activity was at the same level in the mutant strain as in the wild type strain in inositol-free medium, and gene expression was regulated by inositol in a similar manner (Fig. 4), thus indicating that the apparent reduced mitochondrial function in cls1 mutants has no affect on PGS1 gene expression.

Conclusions-- Previous results had established that PG-P synthase activity in yeast was responsive to cross pathway regulation and mitochondrial function. The results presented here establish that the response to carbon source, mitochondrial function, and inositol as well to the catalytic capacity of the cell to synthesize CDP-DAG is at the level of regulation of PGS1 gene expression. Still unresolved is the mechanism by which inositol induces a rapid decline in PG-P synthase activity as compared with the slower response brought about by repression of gene expression. Future experiments will address the cis- and trans-acting elements that regulate PGS1 gene expression in response to carbon source and mitochondrial function.

    ACKNOWLEDGEMENTS

We thank John Lopes for providing plasmid pMA109 and Susan Henry for providing the ino2 and ino4 mutant strains.

    FOOTNOTES

* This work was supported in part by Grant GM54273 from the National Institutes of Health (to W. D.).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.

Dagger Current address: Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702.

§ To whom reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, TX 77225. Tel.: 713-500-6051; Fax: 713-500-0652; E-mail: wdowhan{at}bmb.med.uth.tmc.edu.

1 The abbreviations used are: PG, phosphatidylglycerol; CL, cardiolipin; PG-P, phosphatidylglycerophosphate; CDP-DAG, CDP-diacylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; UASINO, upstream activating sequence responsive to inositol; PPGS1-lacZ, promoter of PGS1 gene placed 5' to the lacZ open reading frame; PGAL1-hCDS, promoter of GAL1 gene placed 5' to the hCDS open reading frame.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
References

  1. Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669[CrossRef][Medline] [Order article via Infotrieve]
  2. Greenberg, M. L., and Lopes, J. M. (1996) Microbiol. Rev. 60, 1-20[Free Full Text]
  3. Chang, S.-C., Heacock, P. N., Clancey, C. J., and Dowhan, W. (1998) J. Biol. Chem. 273, 9829-9836[Abstract/Free Full Text]
  4. Gallet, P. F., Petit, J.-M., Maftah, A., Zachowski, A., and Julien, R. (1997) Biochem. J. 324, 627-634[Medline] [Order article via Infotrieve]
  5. Kuchler, K., Daum, G., and Paltauf, F. (1986) J. Bacteriol. 165, 901-910[Medline] [Order article via Infotrieve]
  6. Zinser, E., Sperka-Gottlieb, C. D., Fasch, E. V., Kohlwein, S. D., Paltauf, F., and Daum, G. (1991) J. Bacteriol. 173, 2026-2034[Medline] [Order article via Infotrieve]
  7. Hoch, F. L. (1992) Biochim. Biophys. Acta 1113, 71-133[Medline] [Order article via Infotrieve]
  8. Abramovitch, D. A., Marsh, D., and Powell, G. L. (1990) Biochim. Biophys. Acta 1020, 34-42[Medline] [Order article via Infotrieve]
  9. Eilers, M., Endo, T., and Schatz, G. (1989) J. Biol. Chem. 264, 2945-2950[Abstract/Free Full Text]
  10. Endo, T., Eilers, M., and Schatz, G. (1989) J. Biol. Chem. 264, 2951-2956[Abstract/Free Full Text]
  11. Snel, M. M., de Kroon, A. I., and Marsh, D. (1995) Biochemistry 34, 3605-3613[Medline] [Order article via Infotrieve]
  12. Tuller, G., Hrastnik, C., Achleitner, G., Schiefthaler, U., Klein, F., and Daum, G. (1998) FEBS Lett. 421, 15-18[CrossRef][Medline] [Order article via Infotrieve]
  13. Jiang, F., Rizavi, H. S., and Greenberg, M. L. (1997) Mol. Microbiol. 26, 481-491[Medline] [Order article via Infotrieve]
  14. Chang, S.-C., Heacock, P. N., Mileykovskaya, E., Voelker, D. R., and Dowhan, W. (1998) J. Biol. Chem. 273, in press
  15. Subik, J. (1974) FEBS Lett. 42, 309-313[CrossRef][Medline] [Order article via Infotrieve]
  16. Greenberg, M. L., Hubbell, S., and Lam, C. (1988) Mol. Cell. Biol. 8, 4773-4779[Medline] [Order article via Infotrieve]
  17. Gaynor, P. M., Hubbell, S., Schmidt, A. J., Lina, R. A., Minskoff, S. A., and Greenberg, M. L. (1991) J. Bacteriol. 173, 6124-6131[Medline] [Order article via Infotrieve]
  18. Janitor, M., and Subik, J. (1993) Curr. Genet. 24, 307-312[Medline] [Order article via Infotrieve]
  19. de Winde, J. H., and Grivell, L. A. (1993) Prog. Nucleic Acids Res. Mol. Biol. 46, 51-91[Medline] [Order article via Infotrieve]
  20. Jakovcic, S. G., Getz, S., Rabinowitz, M., Jacob, H., and Swift, H. (1971) J. Cell Biol. 40, 490-502
  21. Kelly, B. L., and Greenberg, M. L. (1990) Biochim. Biophys. Acta 1046, 144-150[Medline] [Order article via Infotrieve]
  22. Schlame, M., and Greenberg, M. L. (1997) Biochim. Biophys. Acta 1348, 201-206[Medline] [Order article via Infotrieve]
  23. Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19-27[Abstract/Free Full Text]
  24. Van Loon, A. P. G. M., Van Eijk, E., and Grivell, L. A. (1983) EMBO J. 2, 1765-1770[Medline] [Order article via Infotrieve]
  25. Shen, H., Heacock, P. N., Clancey, C. J., and Dowhan, W. (1996) J. Biol. Chem. 271, 789-795[Abstract/Free Full Text]
  26. Anderson, M. S., and Lopes, J. M. (1996) J. Biol. Chem. 271, 26596-26601[Abstract/Free Full Text]
  27. Shen, H., and Dowhan, W. (1997) J. Biol. Chem. 272, 11215-11220[Abstract/Free Full Text]
  28. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Laboratory Course Manual for Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  29. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  30. Stevens, B. (1981) in The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance (Strathern, J. N., Jones, E. W., and Broach, J. R., eds), pp. 471-504, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  31. Olesen, J., Hahn, S., and Guarente, L. (1987) Cell 51, 953-961[Medline] [Order article via Infotrieve]
  32. Forsburg, S. L., and Guarente, L. (1989) Genes Dev. 3, 1166-1178[Abstract]
  33. Kuroda, S., Otaka, S., and Fujisawa, Y. (1994) J. Biol. Chem. 269, 6153-6162[Abstract/Free Full Text]
  34. Klig, L. S., Homann, M. J., Kohlwein, S. D., Kelley, M. J., Henry, S. A., and Carman, G. M. (1988) J. Bacteriol. 170, 1878-1886[Medline] [Order article via Infotrieve]
  35. Shen, H., and Dowhan, W. (1996) J. Biol. Chem. 271, 29043-29048[Abstract/Free Full Text]
  36. Weeks, R., Dowhan, W., Shen, H., N., B., Meengs, B., Nudelman, E., and Leung, D. W. (1997) DNA Cell Biol. 16, 281-289[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.