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The Saccharomyces SUN gene, UTH1, is involved in cell wall biogenesis

James J. Ritch, Shawn M. Davidson, Jared J. Sheehan, Nicanor Austriaco O.P.
DOI: http://dx.doi.org/10.1111/j.1567-1364.2009.00601.x 168-176 First published online: 1 March 2010

Abstract

Deletion of the Saccharomyces gene, UTH1, a founding member of the SUN family of fungal genes, has pleiotropic effects. Several phenotypes of Δuth1 cells including their decreased levels of mitochondrial proteins, their impaired autophagic degradation of mitochondria, and their increased viability in the presence of mammalian BAX, a proapoptotic regulator localized to the mitochondria, have prompted others to propose that the Uth1p functions primarily at the mitochondria. In this report, we show that cells lacking UTH1 have more robust cell walls with higher levels of β-d-glucan that allows them to grow in the presence of calcofluor white or sodium dodecyl sulfate, two reagents known to perturb the yeast cell wall. Moreover, these Δuth1 cells are also significantly more resistant to spheroplast formation induced by zymolyase treatment than their wild-type counterparts. Surprisingly, our data suggest that several of the enhanced growth phenotypes of Δuth1 cells, including their resistance to BAX-mediated toxicity, arise from a strengthened cell wall. Therefore, we propose that Uth1p's role at the cell wall and not at the mitochondria may better explain many of its effects on yeast physiology and programmed cell death.

Keywords
  • UTH1
  • PKC1
  • SUN genes
  • cell wall biogenesis
  • β-d-glucan
  • chitin

Introduction

A founding member of the SUN (SIM1, UTH1, and NCA3) family of fungal genes, UTH1 was originally identified in a genetic screen for Saccharomyces cerevisiae mutants that increased the stress resistance and the replicative lifespan of yeast cells (Kennedy, 1995; Austriaco, 1996). Mutant cells lacking UTH1 have longer replicative lifespans (Kennedy, 1995; Austriaco, 1996); are capable of growing at elevated temperatures (Austriaco, 1996; Camougrand, 2003); are resistant to hydrogen peroxide (Austriaco, 1996; Bandara, 1998) and rapamycin (Camougrand, 2003); and are sensitive to copper (Jo, 2008) and to paraquat (Austriaco, 1996; Bandara, 1998). Finally, Δuth1 cells are also able to grow in the presence of overexpressed proapoptotic mammalian BAX, suggesting that UTH1 may be involved in the regulation of yeast-programmed cell death (Camougrand, 2003).

What does Uth1p do? Several lines of evidence have prompted Camougrand and colleagues to propose that the Uth1p protein functions primarily at the mitochondria. First, they showed that the inactivation of UTH1 reduces the levels of mitochondrial proteins including cytochrome aa3, c, and b, and citrate synthase (Camougrand, 2000). Next, they localized Uth1p to the outer mitochondrial membrane and to the cell wall (Velours, 2002). Third, as we have already noted above, they observed that deleting UTH1 allows yeast cells to survive the overexpression of mammalian BAX, a proapoptotic protein known to act at yeast mitochondria (Camougrand, 2003). Finally, they discovered that the autophagic degradation of mitochondria is impaired in Δuth1 cells (Kissova, 2004, 2007). In light of these data, Camougrand (2004) have suggested that Uth1p is a regulator of mitochondrial function and that this role may explain its diverse effects on yeast apoptosis and cell physiology.

In this paper, we report that UTH1 is involved in cell wall biogenesis: cells lacking UTH1 have more robust cell walls that are relatively more resistant to enzymatic attack by zymolyase than their wild-type counterparts, probably because they contain higher levels of β-d-glucan. Surprisingly, our data also suggest that several of the enhanced growth phenotypes of Δuth1 cells, including their resistance to BAX-mediated toxicity, arise from a strengthened cell wall. Therefore, we propose that Uth1p's role at the cell wall and not at the mitochondria may better explain many of its effects on yeast physiology and programmed cell death.

Materials and methods

Yeast strains, plasmids, and growth conditions

All experiments were performed with isogenic strains in the W303-1A background (MATa ade2, his3, leu2, trp1, ura3, ssd1-d2). The Δuth1 mutant was created by disrupting the ORF with the kanR marker using a PCR-based knockout strategy (Brachmann, 1998) and verified both by PCR and by phenotypic analysis. To overexpress either human BAX or yeast PKC1 in our strains, we transformed either plasmid pCM189/Bax (Camougrand, 2003) or plasmid pFR22 (Roelants, 2004) into our cells and plated them on selective media. Doxycycline supplementation was used to regulate BAX expression as described previously (Camougrand, 2003). For all the experiments described in this paper, cells were cultured using standard protocols (Amberg, 2005), and transformations were accomplished using the lithium acetate method (Gietz & Schiestl, 2007). Unless noted otherwise, all drugs were purchased from Sigma-Aldrich.

Spot assays

Cells were grown overnight in either rich or selective media at 30 °C and then diluted to a final concentration (an approximate OD660 nm value of 0.2). For each strain, a series of 10-fold dilutions was then prepared in water over a range of concentrations from 10−1 to 10−5 relative to the initial culture. Spots of 5 μL from each dilution series were then plated on the indicated media and cultured at either 30 or 39 °C for 2, 3, or 5 days, depending on the particular plate. Plates supplemented with drugs were poured and used on the same day. All spot assays were repeated at least three times and a representative experiment is shown.

Spheroplast rate assay

Enzyme preparation and cell wall lysis assay were based on the method described previously (Ovalle, 1998). Briefly, cells were grown overnight in rich media at 30 °C, harvested, and washed three times with deionized water, and resuspended to an OD660 nm of 0.5 in TE buffer, pH 7.5 (50 mM Tris/HCl, 5 mM EDTA). Zymolyase (5 U μL−1; Zymo Research, Orange, CA) was then added to the cells to a final concentration of 12 μg mL−1. Cell suspensions were incubated at 23 °C and their OD was recorded at 4-min intervals for the indicated time period.

Analysis of cell wall sugar composition

Chitin levels were quantified according to a method described previously (Lesage, 2005). Yeast cultures were first grown to the stationary phase in liquid yeast peptone dextrose (YPD) medium and then diluted 1 : 100 in fresh YPD and incubated at 30 °C with shaking for 18–22 h. Typically, 1 mL of culture was spun in a microfuge tube at 14 K for 2 min and the media were removed. The cell pellets were then air dried at 37 °C for 2–3 days. Next, cell pellets were resuspended in 1 mL of 6% KOH and heated at 80 °C for 90 min with occasional mixing. Alkaline-insoluble material was pelleted (20 000 g, 20 min) and neutralized with phosphate-buffered saline for 10–20 min with occasional mixing. After centrifugation (20 000 g, 20 min), 200 μL of McIlvaine's Buffer (0.2 M Na2HPO4/0.1 M citric acid, pH 6.0) was added to the pellets. Extracts were then stored at −20 °C until processed for chitin measurements. Samples were thawed and subjected to digestion with 5 μL of 5 mg mL−1 chitinase from Trichoderma viride (Sigma-Aldrich) at 37 °C for 36–40 h and then for 20–24 h. The amounts of chitin were then determined using a modified Morgan–Elson procedure as described previously (Bulik, 2003). The levels of chitin, expressed as GlcNAc concentrations, were then normalized to the dry weight of the sample.

Next, β-d-glucan levels were quantified according to the methods of Boone (1990) and Yiannikouris (2004). Yeast cells were grown as 10-mL cultures of YPD until the stationary phase. Cells were harvested, washed once with distilled water, and then extracted three times with 0.5 mL of 3% NaOH at 75 °C (1 h per extraction). After alkali extraction, the alkali-insoluble material was washed once with 1 mL of 100 mM Tris-HCl (pH 7.5) and once with 1 mL of 10 mM Tris-HCl (pH 7.5). The washed residue was then digested for 16 h at 37 °C with 1 mg of Zymolyase 100T (United States Biological, Swampscott, MA) in 1 mL of 10 mM Tris-HCl (pH 7.5). The insoluble pellet that remains after zymolyase digestion was removed by centrifugation, and the supernatant was dialyzed against distilled water, using Spectra/por tubing with a 6000–8000-D pore size (Spectrum Laboratories, Rancho Dominguez, CA), for 16 h. The carbohydrate content before dialysis [(1,3) plus (1,6) β-glucan] and post dialysis [(1,6) β-glucan alone] was measured as hexose using the phenol–sulfuric acid method (Dubois, 1956). The levels of β-d-glucan were then normalized to the dry weight of the sample. Finally, supernatants obtained from the alkali extraction containing alkali soluble β-glucans and mannoproteins from yeast cell walls were dialyzed (1 : 100, v/v) with 0.02 M Tris-HCl buffer (pH 7.4) for at least 16 h at 4 °C with 0.02 M Tris-HCl buffer (pH 7.4). Mannans and β-glucans were separated on a concanavalin A Sepharose column (Pharmacia) at 4 °C as described previously (Yiannikouris, 2004). Alkali-soluble β-glucans were eluted with 0.02 M Tris-HCl buffer (pH 7.4)/0.5 M NaCl and stored at −20 °C until the carbohydrate content was measured as above, using the phenol–sulfuric acid method.

Results and discussion

Deleting UTH1 has pleiotropic effects including phenotypes associated with the endoplasmic reticulum (ER)

Deletion of the gene, UTH1, has pleiotropic effects. As we and others have shown previously, mutant cells lacking UTH1 are capable of growing at elevated temperatures; are resistant to hydrogen peroxide; and are sensitive to copper and to paraquat (Fig. 1). In addition, we have also discovered that they are resistant to β-mercaptoethanol (Fig. 1) and to dithiothreitol (data not shown), drugs known to induce ER stress (Cox & Walter, 1996). All of these phenotypes suggest that UTH1 function involves numerous physiological processes in the cell.

1

UTH1 is involved in the yeast cell's response to different stresses. Aliquots (5 μL) of 10-fold serial dilutions of wild-type and Δuth1 mutant cells in the W303-1A strain background were plated onto the designated media and cultured at either 30 or 39°C for 2, 3, or 5 days, depending on the particular plate. All spot assays were repeated at least three times, and a representative experiment is shown.

UTH1 is involved in maintaining the integrity of the yeast cell wall

UTH1 is a founding member of the SUN family of fungal genes (Austriaco, 1996). Four other fungal SUN genes, SUN4 in S. cerevisiae (Mouassite, 2000), psu1 in Saccharomyces pombe (Omi, 2005), and SUN41 and SIM1/SUN42 in Candida albicans (Firon, 2007; Hiller, 2007; Sosinska, 2008), have been implicated in the regulation of the integrity of the yeast cell wall. To test whether UTH1 has a similar function, we plated wild-type and Δuth1 cells on media containing either calcofluor white (CFW) or sodium dodecyl sulfate (SDS), two reagents known to perturb the yeast cell wall (Kaeberlein & Guarente, 2002), and observed that the mutant is more resistant to these agents than the wild-type strain (Fig. 2a). This suggested that deleting UTH1 strengthens the yeast cell wall.

2

UTH1 is involved in maintaining the integrity of the yeast cell wall. (a) Deleting UTH1 enhances cell growth on media supplemented with CFW or SDS. Aliquots (5 μL) of 10-fold serial dilutions of wild-type and Δuth1 mutant cells in the W303-1A strain background were plated onto the designated media and cultured at 30°C for 2 or 4 days, depending on the particular plate. YPDS denotes YPD plates supplemented with 1 M sorbitol, respectively. (b) Overexpression of PKC1 enhances cell wall integrity, mimicking a deletion of UTH1. Aliquots (5 μL) of 10-fold serial dilutions of wild-type and Δuth1 mutant cells transformed with plasmid pFR22 to overexpress PKC1 were plated onto the designated selective synthetic dextrose (SD) plates and cultured at 30°C for 2 or 5 days. SD-S denotes SD plates supplemented with 1 M sorbitol. All spot assays were repeated at least three times, and a representative experiment is shown.

To further characterize this phenotype, we plated wild-type and Δuth1 cells on CFW and on SDS plates supplemented with 1 M sorbitol, which provides osmotic stabilization and prevents lysis caused by a weakened cell wall (Kaeberlein & Guarente, 2002). Under these conditions, there is no difference in the growth of our wild-type and mutant strains, suggesting that the deletion of UTH1 phenocopies the supplementation of growth media with an osmotic stabilizer (Fig. 2a).

To confirm our findings, we overexpressed PKC1 in wild-type and Δuth1 cells using a high-copy plasmid and plated them on SDS plates with or without osmotic stabilization. Pkc1p is a central integrator of cell integrity that acts to promote transcription of cell wall biosynthetic genes (Heinisch, 1999). We observed that the growth rates of wild-type cells, wild-type cells overexpressing PKC1, and Δuth1 cells are indistinguishable on SDS plates supplemented with sorbitol, suggesting that deleting UTH1 and overexpressing PKC1 have the same effect of enhancing cell wall integrity (Fig. 2b). Because we see no difference in growth between wild-type cells overexpressing PKC1, wild-type cells on sorbitol, and wild-type cells overexpressing PKC1 on sorbitol, on SDS plates, our data suggest that the phenotypes we observe from either of these manipulations – either the overexpression of PKC1 or the supplementation of media with 1 M sorbitol – can be attributed directly to their enhancement of cell wall integrity rather than to an indirect effect caused either by a non-cell-wall-related function of overexpressed PKC1 (Fairn, 2007) or by the sorbitol-mediated activation of the osmoregulatory high osmolarity glycerol pathway (Saito & Tatebayashi, 2004). Otherwise, we would have observed a synergistic effect on the growth of wild-type cells overexpressing PKC1 on SDS plates supplemented with sorbitol.

Finally, to more directly assay the structural integrity of the wild-type and Δuth1 yeast cell walls, we compared the rates of formation of spheroplasts of wild-type and mutant Δuth1 yeast cells cultured in a hypotonic solution in the presence of zymolyase, a mixture of cell wall-digesting enzymes (Ovalle, 1998). This spheroplast rate assay has been used by others to show that Acb1p (Gaigg, 2001), Pho85p (Huang, 2002), and Bet1p (Kipnis, 2004) are all involved in maintaining the integrity of the yeast cell wall. As shown in Fig. 3, mutant cells lacking UTH1 were significantly more resistant to zymolyase treatment than wild-type cells. More specifically, for wild-type cells, the mean lag time (LT) in the presence of zymolyase for three independent samples – where LT has been estimated by interpolation of the lysis curve as the time in which the OD660 nm declined by 0.05 from its initial value (Ovalle, 1998) – was 12 min and the mean maximal lysis rate (MLR) was 0.230 – where MLR has been defined as the absolute value of the slope of the least-squares fit line for the portion of the lysis curve with the steepest log-linear decline (Ovalle, 1998). In contrast, for the Δuth1 mutants, the mean LT for three independent samples was 18.6 min and the MLR was 0.074. The rate indices (RI) of the wild-type and Δuth1 cells, where RI has been defined as MLR/LT (Ovalle, 1998), were 0.0192 and 0.0041, respectively (P<0.002). These results demonstrate that the walls of Δuth1 cells are more resistant to enzymatic attack than those of wild-type controls, confirming the findings from the genetic analysis that had suggested that the cell walls of Δuth1 cells are more robust.

3

Deleting UTH1 decreases the rate of spheroplast formation of yeast cells. Cells of the indicated genotypes were cultured overnight in rich YPD media at 30°C, harvested, and washed three times with deionized water, and then resuspended to an OD660 nm of 0.5 in TE buffer, pH 7.5 (50 mM Tris/HCl, 5 mM EDTA). Zymolyase (5 U μL−1; Zymo Research) was then added to the cells to a final concentration of 12 μg mL−1. Cell suspensions were incubated at 23°C and their OD660 nm was recorded at 4-min intervals for the indicated time period. Assays were performed in triplicate, and the mean values are shown along with the standard deviation (SD).

Taken together, both our genetic and biochemical data suggest that UTH1's function parallels the role of four other fungal SUN genes: psu1, which is involved in cell wall synthesis in S. pombe (Omi, 2005); SUN4, which is involved in cell septation and that, significantly, appears to act in concert with UTH1 (Mouassite, 2000); and SUN41 and SIM1/SUN42, which have been implicated in cell wall remodeling in C. albicans (Firon, 2007). In some way, UTH1 is involved in the biogenesis of the cell wall in S. cerevisiae.

Deleting UTH1 alters the polysaccharide composition of the yeast cell wall

How does UTH1 regulate the integrity of the yeast cell wall? Firon (2007) have shown that UTH1 is able to complement a sun41ΔΔsun42Δ double mutant in C. albicans. Given that SUN41 and SIM1/SUN42 mutants manifest specific cell wall defects at the septa in Candida and that these Candida mutants are sensitive only to cell wall-perturbing agents that are specific to chitin synthesis, these authors have proposed that UTH1 is involved in chitin biosynthesis in S. cerevisiae. To test this hypothesis, we determined the chitin levels in the cell walls of Δuth1 cells and showed that they indeed have decreased amounts of chitin as compared with wild-type controls (Table 1).

View this table:
1

Chitin and glucan composition of the cell wall

Chitin Total chitin (nmol GlcNAc mg−1 dry weight)Glucan
Alkaline insolubleAlkaline soluble
Total β-glucan (μg g−1 dry weight)(1,6) β-glucan (μg g−1 dry weight)Total β-glucan (μg g−1 dry weight)
W303-1A21.3 (1.75)190 (28)51 (8)21 (44)
Δuth115.9 (1.34)488 (52)90 (22)247 (109)
(P<0.002)(P<0.001)(P<0.05)(P<0.05)
  • Deleting UTH1 alters the polysaccharide content of the yeast cell wall. Chitin and β-d-glucan levels were quantified as described in Materials and methods. Assays were performed in triplicate, and the mean values are shown along with the standard deviation (SD) in parentheses. Statistical significance was calculated using Student's unpaired t-test.

This finding – that Δuth1 cells have lower chitin levels – was unexpected. As shown in Figs 2 and 3, Saccharomyces cells lacking UTH1 are not only more resistant to CFW and to SDS, two reagents that are known to destabilize the cell wall (Kaeberlein & Guarente, 2002), but are also significantly more resistant to zymolyase, a mixture of cell wall-digesting enzymes (Kitamura & Yamamoto, 1972). This would not be expected if deleting UTH1 only reduces the chitin content of the cell wall, an effect expected to weaken and not to strengthen the cell wall. Interestingly, one previous report has shown that mutant cells with increased resistance to CFW have lower chitin levels than their wild-type counterparts (Roncero, 1988).

How could lower levels of chitin lead to the strengthening of the yeast cell wall? Some have suggested that a compensatory mechanism exists in yeast in response to cell wall damage, whereby decreases in β-d-glucan levels lead to a compensatory increase in chitin levels (Kapteyn, 1997; Popolo, 1997; Ram, 1998; Valdivieso, 2000). To explain our data, therefore, we predicted that the reverse mechanism may also exist: the lower levels of chitin in Δuth1 cells may be accompanied by compensatory higher levels of β-d-glucan. To test this hypothesis, we determined the β-d-glucan levels of Δuth1 cells. Indeed, as predicted, these mutant Δuth1 cells had cell walls with significantly higher levels both of total alkaline-soluble and total alkaline-insoluble β-d-glucan and of alkaline-insoluble (1,6) β-d-glucan more specifically (Table 1). This would explain why Δuth1 cells are more resistant to zymolyase, a mixture of cell wall-digesting enzymes composed primarily of β-1,3-glucan laminaripentaohydrolase and β-1,3-glucanase (Kitamura & Yamamoto, 1972). The higher levels of β-d-glucan would also explain our genetic data that had suggested that the cell walls of Δuth1 cells are more robust than their wild-type counterparts.

In sum, our data suggest that UTH1 is involved in the biogenesis of the yeast cell wall. The precise molecular mechanism behind this role, however, remains unknown: Uth1p could be involved in any of the regulatory pathways that have been linked to the complex process of cell wall assembly in S. cerevisiae (Lesage & Bussey, 2006). It is significant that the Uth1p homolog in C. albicans, Sim1p, has been identified as a covalently linked cell wall protein by MS (Sosinska, 2008). Another group has also shown that Uth1p appears to act in concert with another SUN protein, Sun4p, to contribute to cell wall morphogenesis and septation, strongly supporting our proposal that the protein is involved in cell wall biogenesis (Mouassite, 2000). Finally, it is striking that two research teams have shown that UTH1 belongs to a set of Ssd1p-associated mRNAs that is enriched in cell wall related genes (Hogan, 2008; Jansen, 2009).

Deleting UTH1 improves growth under diverse stress conditions by enhancing cell wall integrity

Finally, to determine whether UTH1's role in regulating the integrity of the yeast cell wall could explain any of the pleiotrophic phenotypes of the Δuth1 mutant, we took wild-type and Δuth1 cells and stressed them on media with or without sorbitol supplementation. We discovered that there is no difference in growth between these two strains either at 39 °C, on media containing either β-mercaptoethanol or copper, or with overexpressed mammalian BAX, as long as they are cultured on media with an osmotic stabilizer (Fig. 4a). This suggested that the growth enhancement attributed to the deletion of UTH1 under these conditions could be explained by the gene's role in cell wall biogenesis rather than on mitochondrial function. This was true regardless of whether the BAX cell death assays were performed on sorbitol-supplemented media containing either glucose or glycerol as a carbon source (data not shown). In both cases, the viability of wild-type and Δuth1 cells overexpressing mammalian BAX was indistinguishable.

4

Deleting UTH1 enhances the cell's response to diverse stresses by strengthening its cell wall. (a) Osmotic stabilization with 1 M sorbitol phenocopies a deletion of UTH1. Aliquots (5 μL) of 10-fold serial dilutions of wild-type and Δuth1 mutant cells were plated onto the designated media and cultured at either 30 or 39°C for 2, 3, or 5 days. YPDS and SD-S denote YPD and SD plates supplemented with 1 M sorbitol, respectively. To overexpress human BAX in our strains, we transformed plasmid pCM189/Bax into our cells and plated them on selective SD media with or without doxycycline supplementation as described previously (Camougrand, 2003). (b) Overexpression of PKC1 enhances cell growth at elevated temperatures, mimicking a deletion of UTH1. Aliquots (5 μL) of 10-fold serial dilutions of wild-type and Δuth1 mutant cells transformed with plasmid pFR22 to overexpress PKC1 were plated onto the designated selective SD plates and cultured at 30 or 38°C for 2 or 5 days. All spot assays were repeated at least three times, and a representative experiment is shown.

To confirm this result, we repeated the assay with wild-type and Δuth1 cells, both overexpressing PKC1 on a high-copy plasmid, and observed that the growth rates of wild-type cells, wild-type cells overexpressing PKC1, and Δuth1 cells, at 38 °C, are indistinguishable on plates supplemented with sorbitol, suggesting once again that the enhanced growth phenotype of Δuth1 cells at elevated temperatures is linked to the enhancement of cell wall integrity (Fig. 4b). As before, the absence of a synergistic effect on the growth at 38 °C of wild-type cells overexpressing PKC1 on plates supplemented with sorbitol suggested that the growth enhancement phenotype could be attributed directly to a strengthened cell wall. Finally, parallel results were obtained on copper-supplemented plates, suggesting that the sensitivity of Δuth1 cells to copper is also mediated by the gene's effect on the integrity of the cell wall (data not shown). It would be interesting to determine whether any of the other phenotypes of Δuth1 cells, especially those associated with Uth1p's putative mitochondrial function, can also be linked to its role in regulating the integrity of the yeast cell wall. In light of our findings, it is intriguing to note that two groups have been unable to find the reported link between Uth1p function and mitochondrial autophagy (Kanki, 2009; Okamoto, 2009). However, it is still not clear whether this was due to differences in the strain background. If so, we speculate that Uth1p's differential effects on mitophagy could be linked to the differences in cell wall composition commonly seen among wild-type yeast strains.

In conclusion, we show that cells lacking UTH1 have more robust cell walls that are resistant to zymolyase treatment because they contain higher levels of β-d-glucan. Surprisingly, our data also suggest that several of the enhanced growth phenotypes of Δuth1 cells, including their resistance to BAX-mediated toxicity, arise from a strengthened cell wall. Thus, we propose that Uth1p's role at the cell wall and not at the mitochondria may better explain many of its effects on yeast physiology and programmed cell death.

Authors' contribution

J.J.R. and S.M.D. contributed equally to this work. J.J.R., S.M.D., and N.A. performed the experiments. J.J.S. constructed yeast strains. J.J.R., S.M.D., and N.A. designed experiments, analyzed data, and wrote the manuscript.

Acknowledgements

We thank Mary Blasik, Gina Contuzzi, Kathleen Cornely, David J. Laprade, Kevin R. Murphy, and Charles Specht (University of Massachusetts School of Medicine, Worchester, MA) for technical assistance, and Brian K. Kennedy (University of Washington, Seattle), Stephen Manon (University of Bordeaux), and Jeremy Thorner (University of California, Berkeley) for strains and/or plasmids. We also thank both Gregory Liszt and Leonard Guarente (MIT) and Charles Specht (University of Massachusetts School of Medicine, Worchester, MA) for helpful discussion. Our laboratory is supported by Rhode Island INBRE Grant P20RR016457 from the National Center for Research Resources (NCRR) of the National Institutes of Health (NIH) and a CAFR grant from Providence College.

Footnotes

  • Editor: Ian Dawes

References

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