OUP user menu

The SUN family of Saccharomyces cerevisiae: the double knock-out of UTH1 and SIM1 promotes defects in nucleus migration and increased drug sensitivity

Mariam Mouassite , Martine G. Guérin , Nadine M. Camougrand
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb08887.x 137-141 First published online: 1 January 2000

Abstract

UTH1 and SIM1 are two of four ‘SUN’ genes (SIM1, UTH1, NCA3 and SUN4/SCW3) whose products are involved in different cellular processes such as DNA replication, lifespan, mitochondrial biogenesis or cell septation. UTH1 or SIM1 inactivation did not affect cell growth, shape or nuclear migration, whereas the double null mutant presented phenotypes of numerous binucleate cells and benomyl sensitivity, suggesting that microtubule function could be altered; the uth1Δsim1Δ strain also presented defects which could be related to the Ras/cAMP pathway: pet phenotype, heat shock sensitivity, inability to store glycogen, sensitivity to starvation and failure of spores to germinate. These observations suggested that Uth1p could be involved as a connection step between pathways controlling growth and those controlling division.

Keywords
  • Saccharomyces cerevisiae
  • Nucleus migration
  • Benomyl
  • Caffeine sensitivity

1 Introduction

A new gene family (SUN) of four genes (SIM1, UTH1, NCA3 and SUN4/SCW3) has been found; the remarkable feature of the gene products is that they share a common stretch of 258 amino acids at their carboxy-terminal end presenting 75–85% identity (database searches). The four gene products appear to be involved in very different cellular functions such as aging processes [1], stress response [2], mitochondrial biogenesis [3,4], DNA replication [5] or cell septation [6]. UTH1 belongs to a group of four unlinked genes involved in aging. In Saccharomyces cerevisiae, life span is defined by the number of cell divisions undergone by mother cells before they stop dividing, and mutations in UTH genes result in an increased number of generations of an individual cell [1]. Recently it was found that UTH1 is also involved in the oxidative stress response [2] and our work showed that it participated in the regulation of the biogenesis of mitochondria [4]. SIM1 (start-independent mitosis) participates in nDNA replication regulation since mutant sim11-433, in a strain lacking Clb1-Clb4, undergoes a second additional round of DNA replication without mitosis, apparently due to a decrease in CDK activity [5].

In recent work, we have shown that UTH1 seemed to be involved in pathways requiring NCA3 or SUN4 activity: inactivation of UTH1 in a nca3Δ strain promoted synergistic effects in mitochondrial biogenesis [4]; on the other hand, it increased the penetrance of the septation defect observed in sun4Δ[6]. This paper describes the double knock-out of UTH1 and SIM1 which was found to promote dramatic alterations in the cell, such as the appearance of binucleate cells and defects which could be related to the RAS adenylate cyclase pathway.

2 Materials and methods

2.1 Strains and media

All yeast strains used in this study were derivatives of the standard haploid strain W303-1B (MATα, ade2, his3, trp1, leu2, ura3, can1). The origin of plasmid pRS313, in which a 1-kb SpeI fragment containing only the UTH1 gene was used to construct the uth1::TRP1 gene, and of plasmid pUC18 carrying sim1::LEU2 was described in [4]; they were used to construct null mutants by the one-step gene replacement method of Rothstein [7]. W303-1B/50 (MATα, ade2, his3, trp1, leu2, ura3, can1) is a rho0 strain given by P.P. Slonimski (Gif/Yvette, France).

Cells were grown on a semi-synthetic medium (SC) (0.67% yeast nitrogen base without amino acids) supplemented with adequate amino acids, depending on the strain studied. Carbon sources are given in the figure legends. Fermentative conditions were obtained by adding (2 mg l−1) antimycin A to the medium supplemented with glucose. Heat shock resistance was determined according to Kennedy et al. [1]. Benomyl sensitivity was tested on SCD plates supplemented with benomyl dissolved in 1% DMSO as described by DeZwaan et al. [8]. Caffeine sensitivity was tested on a range of concentrations (6–12 mM caffeine) at 28°C [9]. Glycogen was visualized according to Toda et al. [10].

2.2 Flow cytometric analysis of DNA

Cells were fixed in 70% ethanol and then treated with RNase and protease as previously described [11], and stained with propidium iodide for analysis on a Coulter EPICS.

2.3 Microscopy

Microscopy was carried out on cells grown on a semi-synthetic medium supplemented with glucose. Cells were fixed by adding formaldehyde directly to the medium to a final concentration of 3.7%. Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI).

3 Results

We first attempted to obtain the double null mutant strain by crossing the two single null mutants uth1Δ and sim1Δ. Diploids were obtained, thus showing no mating deficiency, which were able to sporulate. Twelve tetrads were examined: eight contained three viable spores, three contained two viable spores and one contained the parental ditypes. However, none of the viable spores contained the two inactivation markers and from this result it can be concluded that the two mutations likely impaired germination of yeast spores or spore viability. The double null mutant was obtained by inactivation of SIM1 in a uth1Δ strain and ascertained using classical methods. The double mutant was complemented by a galactose-inducible plasmid containing either UTH1 or SIM1; defects observed in the uth1Δsim1Δ strain were rescued in transformants carrying either of these plasmids (see Fig. 4).

4

FACS analysis. Cells were grown on a semi-synthetic medium supplemented with 1% glucose and harvested in exponential growth phase. Wild-type (a), sim1Δ (b), uth1Δ (c), uth1Δsim1Δ (d); uth1Δsim1Δ was transformed with a galactose-inducible plasmid bearing UTH1 grown on glucose (e) or galactose (f).

3.1 Growth parameters

Measurements of growth parameters on glucose showed that the doubling time of the single mutants uth1Δ and sim1Δ was equal to that of wild-type, but was twice as long in the double null uth1Δsim1Δ. The maximal biomass obtained under fermentative conditions (Fig. 1A) was lower than in the case of each of the single null mutants (the higher saturation density reached by uth1Δ has been previously described [4]). The double null mutant was not cold- or thermo-sensitive, but exhibited a very reduced viability in stationary growth phase when kept for 3 days at 28°C (Fig. 1B).

1

Growth and maintenance of viability in wild-type and mutant cells. A: Saturation densities were determined at 28°C on a 0.5% glucose SC medium added with 2 mg l−1 of antimycin. B: Viability. Cells were grown until stationary phase, kept for 3 days at 4°C (a) or at 28°C (b) and spotted with serial dilutions on SCD plates.

As shown in Fig. 2, the uth1Δsim1Δ strain did not grow on galactose or lactate and grew very poorly on raffinose. Crossing uth1Δsim1Δ with W303-1B/50, a ρ0 wild-type strain, resulted in a diploid able to grow on lactate, indicating that the double null mutant was not a ρ strain (not shown). This result also indicated that the double null mutant was able to conjugate. Since the respiratory deficiency of uth1Δsim1Δ was not due to a defect in the mtDNA, cytochrome spectra were analyzed to verify expression of this mtDNA which encodes apocytochrome b and three subunits of cytochrome c oxidase [12]: the presence of cytochromes aa3 and b in the double null mutant grown on glucose indicated that the pet phenotype was not due to a defect in the expression of the mtDNA or in the assembly of these respiratory complexes (not shown).

2

Phenotypes of uth1Δsim1Δ. Cells were harvested in exponential growth phase and washed. 1: W303-1B; 2: uth1Δ; 3: sim1Δ; 4:uth1Δsim1Δ. Serial dilutions were prepared (104 to 102) and cells were spotted on SC plates added with (A) glucose, (B) galactose, (C) raffinose, and (D) lactate. Drug sensitivity was measured on SCD plates supplemented with: (E) 7 mM caffeine, (F) 8 mM caffeine, (G) 10 μg ml−1 benomyl.

3.2 Morphological studies

From microscopic analysis, uth1Δsim1Δ appeared as cells very heterogeneous in size and shape (Fig. 3). However, DNA staining with DAPI showed that numerous cells presented defects in nuclear localization and distribution levels. Several different cases were observed (Fig. 3): (a) some cells were binucleate, the daughter cells being anucleate; (b) cytokinesis failed to complete; (c) despite the presence of a bud, the nucleus remained undivided at the neck. In all cases, mitochondria were normally transmitted, suggesting that microfilaments that organize the vectorial transport of organelles to the daughter cell were not defective. To obtain a better insight, a FACS analysis was carried out on asynchronous culture (Fig. 4): the parental, uth1Δ and sim1Δ mutants showed 1C and 2C DNA peaks. In contrast, with uth1Δsim1Δ additive peaks at more than 2C were observed (Fig. 4d). Expression of UTH1 in the double null mutant restored the correct DNA repartition (Fig. 4f).

3

Microscopic analysis. Nomarski optics and DAPI staining of uth1Δsim1Δ mutant. m: mitochondria; a, b and c denote cells with different phenotypes as described in the text.

Polyploidy can be due to different mechanisms: endomitosis, DNA replication in the absence of karyokinesis and cytokinesis, or alterations in processes required for chromosome segregation and nuclear migration, such as defects in microtubule function or integrity. Benomyl is a microtubule depolymerizing drug, and mutations in genes that regulate microtubule function often result in altered sensitivity to drugs that destabilize microtubules [13]. Strains deleted for TUB3 or KIP2, encoding an α-tubulin and a kinesin respectively, were hypersensitive to benomyl [1416], whereas strains deleted for DYN1 have the same benomyl sensitivity as the wild-type [8,16] and the ones deleted for KIP3 and KAR3, two other kinesin genes, were resistant to the drug [8]. Sensitivity of the null mutants to benomyl was tested by spotting cells on SCD plates containing different drug concentrations. Fig. 2 shows that uth1Δ or sim1Δ presented the same benomyl sensitivity as the wild-type whereas the double null mutant was sensitive to the drug, suggesting decreased microtubule stability in this mutant.

3.3 Is the nuclear migration defect related to a defect in a cellular signalling pathway?

Further analysis of the physiological parameters of uth1Δsim1Δ revealed that, in addition to the inability of this double null mutant spore to germinate and its pet phenotype, it was unable to accumulate glycogen (Fig. 5). Moreover, uth1Δsim1Δ was very sensitive to heat shock, whereas each of the single null mutants exhibited a higher heat shock resistance, a phenotype already described for uth1Δ[1], but not for sim1Δ. Indeed, the percent survival was 28, 77, 62 and 5 for W303-1B, uth1Δ, sim1Δ and uth1Δsim1Δ respectively. Taken all together, simultaneous inactivation of UTH1 and SIM1 resulted in phenotypes similar to that of mutants affected in the Ras-adenylate cyclase pathway required for cell cycle progression and for the heat shock response [17]. These observations prompted us to test the caffeine sensitivity, a drug which is known to interfere in a wide variety of processes, including the progression of the cell cycle [18] and changes in cell morphology [19]. Moreover, the growth sensitivity to caffeine is often associated with defects in components of mitogen-activated protein (MAP) kinase pathways [20]: Fig. 2E,F shows that sim1Δ was slightly sensitive to caffeine and the double null mutant hypersensitive to this drug. This caffeine sensitivity was not rescued by addition of 1 M sorbitol in the growth medium and uth1Δsim1Δ did not exhibit any phenotype associated with osmotic stress (not shown). Growth of uth1Δsim1Δ was unaffected by drugs such as staurosporine, calcofluor or SDS, known to affect the protein kinase C pathway [21]. Thus, the caffeine sensitivity of this mutant was not linked to this pathway, which is a part of a regulatory MAP kinase cascade [22].

5

Glycogen storage. Cells were grown on YPD medium for 3 days (stationary phase) and glycogen was visualized according to Toda et al. [10]. 1: W303-1B; 2: uth1Δ; 3: sim1Δ; 4:uth1Δsim1Δ.

4 Discussion

In this paper we described the cellular catastrophe resulting from the double inactivation of two genes of the SUN family, UTH1 and SIM1. Neither of the single null mutants displayed any defect related to the phenotypes of the double null mutant.

It has already been reported that inactivation of UTH1 promotes pleiotropic effects at different cellular levels, such as aging [1], stress resistance [1,2] and mitochondrial biogenesis [4]. SIM1 was described as a gene involved in DNA replication since mutation of this gene in a clb1-4ts strain allowed the cell to replicate its DNA without mitosis and to re-bud; however, the sim1-433 mutant contained a single undivided nucleus [5]. Concerning SIM1, two observations have to be taken in account: (i) in a wild-type strain, inactivation of SIM1 was without any apparent consequence; (ii) the promoter sequence of SIM1 contains a SCB regulation box [23] at −300 bp indicating that the expression of this gene could be cell cycle-regulated: the dramatic effect of mutation in SIM1 observed in a strain deleted for cyclins 1–4 could result in a deregulation of the cell cycle. As a matter of fact, overexpression of ClB5 was able to rescue the defect [5].

In the uth1Δsim1Δ strain, we observed numerous binucleate cells and also benomyl sensitivity, indicating that in this mutant microtubules could be less stable to depolymerization than in wild-type. In addition to the nuclear migration defect, numerous pleiotropic phenotypes (sensitivity to heat shock, low levels of glycogen storage, reduced viability, inability to grow on non-fermentable carbon sources and failure of spores to germinate) were observed in the double null mutant which could be related to a perturbation in the Ras-adenylate cyclase pathway [17].

From previous work, it appeared that Uthp, in addition to its involvement in aging [1] and oxidative stress response [2], participated in three signaling functions: the first, shared with NCA3p, was required for mitochondrial biogenesis [4]; the second, shared with Sun4p, was involved in cell separation [6]; the third, described in this paper, interfered with Sim1p in such a way that Uth1p was essential for normal growth and cell division, since cell depletion in Uth1p and Sim1p generated phenotypes mimicking a hyperactivation of the Ras-adenylate cyclase pathway [17]. The activity of this pathway in S. cerevisiae is strictly controlled and the connection between the molecules controlling growth (Ras/cAMP) and those controlling division (cyclins) explained how division is co-ordinated with growth [24]. Further investigations should allow us to determine the cellular roles of Uth1p and Sim1p in order to understand the mechanisms by which these proteins act on these signalling pathways.

Acknowledgments

The authors wish to thank G. Demaison for her excellent technical assistance, Dr. Belloc (Hopital du Haut Levêque, Pessac) for welcoming us to use the Coulter EPICS flow cytometry system and Dr. M. Bonneu for his help in fluorescence microscopy. This work was supported by grants from the Victor Segalen University of Bordeaux II, The Centre National de la Recherche Scientifique, the Conseil Régional d'Aquitaine and the Association pour la Recherche contre le Cancer.

References

  1. [1]
  2. [2]
  3. [3]
  4. [4]
  5. [5]
  6. [6]
  7. [7]
  8. [8]
  9. [9]
  10. [10]
  11. [11]
  12. [12]
  13. [13]
  14. [14]
  15. [15]
  16. [16]
  17. [17]
  18. [18]
  19. [19]
  20. [20]
  21. [21]
  22. [22]
  23. [23]
  24. [24]
View Abstract