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KEM1 is involved in filamentous growth of Saccharomyces cerevisiae

Jaehee Kim, Jinmi Kim
DOI: http://dx.doi.org/10.1111/j.1574-6968.2002.tb11410.x 33-38 First published online: 1 October 2002

Abstract

The KEM1/XRN1 gene was originally identified because of its functions in microtubule-mediated processes, and is also known to be a major cytoplasmic 5′-3′ exoribonuclease gene, which is involved in RNA turnover. Here we present evidence that KEM1 plays a role in filamentous growth. In Saccharomyces cerevisiae, the filamentation signalling shares multiple components of the MAP kinase cascade (STE7; STE11, and KSS1) and the transcription factor STE12 with mating process. Both haploid invasive growth and diploid pseudohyphal growth were found to be greatly impaired in kem1 mutant strains. KEM1 affected the level of FLO11 transcripts and the expression of the filamentation-associated reporter genes; Ty1-lacZ and FLO11-lacZ. Suppression analysis implies that KEM1 does not affect the RAS/PKA pathway, but that it possibly functions downstream of the MAP kinase pathway during filamentation.

Keywords
  • KEM1
  • Filamentation
  • Nuclear fusion
  • Exoribonuclease
  • XRN1
  • MAP kinase

1 Introduction

KEM1 was initially identified because of its functions in microtubule-mediated processes in Saccharomyces cerevisiae [1]. Mutations in KEM1 cause a nuclear fusion defect during mating, increased sensitivity to microtubule-destabilizing drug benomyl, and defects in chromosome transmission, spindle pole body separation, and nuclear migration during mitosis. The Kem1 protein was subsequently reported to be a microtubule-associated protein [2]. Purified Kem1p promoted in vitro assembly of tubulin into microtubules.

Other groups have also shown that Kem1p (= Xrn1p) is a major cytoplasmic 5′-3′ exoribonuclease and is responsible for mRNA turnover [35]. In kem1 (=xrn1) mutant cells, 5′-3′ degradation of stable PGK1 mRNA or unstable MFA2 mRNA tends to slow down, and overall cellular mRNA and protein levels are higher in kem1 mutants [4,6,7]. The exonuclease motifs present in Kem1p are significant in the exonuclease activity and the benomyl phenotype of Kem1p [8]. In addition, the nuclear exoribonuclease, Rat1p, is interchangeable with Kem1p in the RNA turnover and the benomyl phenotypes [3,9]. However, the mutant Kem1p lacking the exoribonuclease activity still showed the microtubule-assembly activity as well as the meiosis-specific functions such as nucleic acid binding and homologous pairing activity [8]. With these diverse biochemical activities, Kem1p is suggested to be a multifunctional protein involved in a number of cellular processes.

Previous studies have shown that kem1 mutant cells lose viability upon prolonged incubation in medium lacking nitrogen [1]. In this study, we asked whether KEM1 plays any role in the filamentation growth of the Σ1278b strains. When starved for nitrogen, diploid yeast cells in the Σ1278b background undergo a dimorphic transition to form pseudohyphae [10]. A related phenomenon, invasive growth, occurs in haploid cells on rich medium [11]. Both haploid invasive growth and diploid pseudohyphal growth were examined in kem1 mutant strains. To demonstrate the functional significance of KEM1 in filamentation, we analyzed the expression of filamentation-specific genes in kem1 strains and also carried out the suppression analysis of kem1 with the components of the RAS/PKA and the MAP kinase pathways.

2 Materials and methods

2.1 Strains, plasmids, and growth conditions

All the S. cerevisiae strains and plasmids used in this study are listed in Table 1. The kem1::URA3 or the kem1::LEU2 mutation was introduced with a linear DNA fragment carrying kem1::URA3 (pJI112) or kem1::LEU2 (pJI113) construct [1]. All deletion mutations were confirmed by Southern blot or PCR analysis. Diploid strain JK353 (KEM1/KEM1) or JK354 (kem1/kem1) was constructed by a cross of 10560-2B and 10560-5B or a cross of JK351 and JK352, respectively.

View this table:
Table 1

Strains and plasmids used in this study

GenotypeSource/reference
Strains
10560-2BMATa ura3-52 his3::hisG leu2::hisGG.R. Fink
10560-5BMATα ura3-52 trp1::hisG leu2::hisGG.R. Fink
JK351MATa ura3-52 his3::hisG leu2::hisG kem1::LEU2This work
JK352MATα ura3-52 trp1::hisG leu2::hisG kem1::LEU2This work
JK353MATa/MATα ura3-52/ura3-52 his3::hisG/+trp1::hisG/+leu2::hisG/leu2::hisGThis work
JK354MATa/MATα ura3-52/ura3-52 his3::hisG/+trp1::hisG/+leu2::hisG/leu2::hisG kem1::LEU2/kem1::LEU2This work
JK357MATa ura3-52 his3::hisG leu2::hisG kem1::URA3This work
Plasmids
pJI74KEM1 URA3 CEN[1]
pJI112kem1::URA3 URA3 CEN[1]
pJI113kem1::LEU2 URA3 CEN[1]
pRS313-KEM12µ ARS HIS3 KEM1This work
YEp355-FLO11::lacZFLO11::lacZ URA3 2µ[20]
pIL30u-URA3Ty1-lacZ URA3 CEN[21]
BHM275FRE(TEC1)::lacZ URA3 2µ[10]
pHL132flo8::lacZ URA3 2µ[22]
YCp50-RAS2Val19RAS2V19 URA3 CEN[23]
pSL1509ste11–4 URA3 CEN[23]
pYBS101STE12 URA3 2µG.R. Fink
pRS202-TEC1TEC1 URA3 2µ[24]
pYSL13FLO11 URA3 2µ[17]
pHL135FLO8 URA3 2µ[22]
YEp-TPK2TPK2 LEU2 2µ[25]
pRS3162µ ARS URA3[26]
pRS4252µ ARS LEU2[27]
pRS4262µ ARS URA3[27]
  • All yeast strains are Σ1278b background.

E. coli and yeast media were prepared using the established procedure [12,13]. Synthetic low ammonium medium (SLAD) was prepared essentially as described [14]. YEPD medium was used for invasive growth tests as previously described [11]. Standard methods of yeast transformation and genetic crosses were used for all strain constructions [12].

2.2 β-Galactosidase assays

β-Galactosidase assays were performed with exponentially growing cultures (OD600= 0.5–1.0) as previously described [15]. The β-galactosidase unit equals (OD420×1000)/(OD600×volume (µl) of culture×minutes of assay).

2.3 Northern blot analysis

Total RNA was prepared as previously described [16]. Twenty micrograms of total yeast RNA was fractionated by electrophoresis through 1.0% formaldehyde gel and transferred to a Nytran membrane (Hoefer). Blottings were performed as described by Sambrook et al.[13]. Probes were labeled with a random primer kit (Amersham). A PCR product corresponding to the FLO11 open reading frame (ORF), 3540-4073, was used as the FLO11 probe. The STE12 or TEC 1 probe corresponds to the STE12 ORF, 1096-1760, or the TEC1 ORF, 600-1351, respectively. A 716-bp PCR product of ACT1 ORF was used as a loading control.

3 Results and discussion

3.1 KEM1 is involved in pseudohyphal and invasive growth

To investigate whether KEM1 influences pseudohyphal growth in response to nitrogen starvation, we deleted this gene in the Σ1278b strain commonly used for filamentation growth studies. Isogenic KEM1/KEM1 wild-type and kem1/kem1 mutant strains were tested for pseudohyphal growth on SLAD [14]. As shown in Fig. 1A, pseudohyphal development was completely absent in the kem1/kem1 diploid strain. This pseudohyphal defect was restored by the introduction of the wild-type KEM1 gene on a CEN-plasmid. Many mutants defective in terms of pseudohyphal growth also show impaired haploid invasive growth in rich solid medium [11]. The haploid kem1 mutant was also defective in invasive growth (Fig. 1B).

Figure 1

Role of KEM1 in invasive and filamentation growth. A: Pseudohyphal growth of isogenic KEM1/KEM1 (JK353) and kem1/kem1 (JK354) diploid strains tested on SLAD media. Diploid kem1/kem1 strain harboring YCp50-KEM1 (pJI74) is also shown. After 5 days of incubation, the colony morphology was photographed. B: Haploid invasive growth of isogenic KEM1 (10560-2B) and kem1 (JK351) strains tested on YEPD. The kem1 mutant strain harboring YCp50-KEM1 (pJI74) is also shown. After 3 days of incubation, the plate was photographed before and after washing the cells off the agar surface.

The expression of several genes, including FLO11 and Ty1-lacZ, are associated with filamentation or invasive growth [10,17]. We examined whether the kem1 mutation affects the expression of any of these filamentation-related genes (Fig. 2A). In haploids, lacZ reporter gene expression of FLO11-lacZ and Ty1-lacZ are greatly reduced in kem1 mutant strains, whereas the expressions of FRE(TEC1)-lacZ and FLO8-lacZ are not much affected by kem1.

Figure 2

Expression levels of filamentation-specific genes in KEM1 and kem1 haploid strains. A: The β-galactosidase activities of plasmids FLO11-lacZ, Ty1-lacZ, FRE(TEC1)-lacZ, or FLO8-lacZ were measured in KEM1 (10560-2B) and kem1 (JK351) strains. The kem1 mutant strain (JK351) harboring pRS313 (HIS3)-KEM1 is also shown as a control. Units are as described in Section 2. B: Northern analysis of FLO11, TEC1, and STE12 transcripts in KEM1 (10560-2B), kem1 (JK351), and kem1/pKEM1 (JK351 with pJI74) strains.

The effect of kem1 on FLO11-lacZ gene expression correlated well with the filamentation defective phenotypes of kem1. The normality of FRE(TEC1)-lacZ and FLO8-lacZ expression in kem1 cells indicates that the kem1 mutation appears not to disturb the general expression of the filamentation-specific genes. Since the expression of FRE(TEC1)-lacZ is also under the regulation of the filamentation-specific MAP kinase signaling pathway, it would appear that the kem1 mutation does not disturb the upstream components of the MAP kinase cascade. Rather, KEM1 may function downstream of STE12/TEC1.

Northern hybridization of wild-type and kem1 transcripts further confirmed this suggestion. As shown in Fig. 2B, the FLO11 transcripts are greatly diminished in the kem1 haploid strain. This effect of kem1 was not observed in the TEC1 transcript level. Rather, TEC1 transcripts were slightly accumulated in kem1 mutant cells, consistent with the previously reported phenotype of kem1(=xrn1) [4,6,7]. The level of STE12 transcript, which reflects the activities of the MAP kinase components, is also not much affected by the presence of the kem1 mutation.

3.2 KEM1 functions downstream of the MAP kinase signaling pathway

Several elements of the mitogen-activated protein (MAP) kinase pathway and cAMP-dependent protein kinase pathway are required for filamentation and invasion [11,18,19]. To identify relationships between KEM1 and these signaling pathways, we analyzed the effect of MAP kinase or PKA stimulation in kem1 mutants. The dominant hyperactive alleles, RAS2val19 or STE11–4, or overexpression plasmids of TEC1, FLO8, TPK2, or STE12 were introduced into kem1 mutants and the filamentation phenotypes observed (Fig. 3). The invasive growth defect of the haploid kem1 mutant was fully restored by the RAS2val19 allele, or by a 2µ-plasmid-carrying TPK2 or FLO8. The diploid filamentation defect was also partially suppressed by RAS2val19, and weakly suppressed by FLO8 overexpression. However, either the STE11–4 allele or STE12 overexpression, both of which normally upregulate MAP kinase signalling, failed to suppress the filamentation defect of the kem1/kem1 strain. In addition, the overexpression of TEC1 did not restore the filamentation phenotype in kem1/kem1 diploid strains.

Figure 3

Suppression analysis of the filamentation phenotypes of kem1. A: Invasive growth test of haploid wild-type (10560-2B), kem1 (JK351), kem1 strain (JK351) carrying YCp50-KEM1 (pJI74), RAS2val19 (YCp50-RAS2val19), STE11–4 (pSL1509), TEC1 (pRS202-TEC1), FLO8 (pHL135), or kem1 strain (JK357) carrying TPK2 (YEp-TPK2) plasmids are shown. After incubating for 3 days, the plate was photographed before and after washing the cells off the agar surface. B: Pseudohyphal growth test of diploid wild-type KEM1/KEM1 (JK353), kem1/kem1 (JK354) containing pRS426 (vector), YCp-KEM1 (pJI74), RAS2val19 (YCp50-RAS2val19), FLO8 (pHL135), STE11–4 (pSL1509), STE12 (pYBS101), or TEC1 (pRS202-TEC1) plasmids. After incubating for 5 days on SLAD media, the colony morphologies (upper panel) were photographed. Lower panels are of colony morphologies after washing. C: Northern analysis of FLO11 transcripts in strains used in A.

The suppression phenotypes were also analyzed at the level of the FLO11 transcript levels (Fig. 3C). The RAS2val19 allele and FLO8 overexpression, but not the STE11–4 allele, restored FLO11 transcription in kem1 cells. These suppression analyses indicate that hyperactivation of the RAS/PKA cascade can bypass the KEM1-dependent pathway during filamentation, and that filamentation activation by the overexpression of STE12 or the hyperactive allele STE11–4 was blocked by the kem1 mutation. We postulate that KEM1 possibly functions downstream of the MAP kinase pathway during filamentation.

Acknowledgements

We are grateful to G.R. Fink for kindly supplying plasmids and strains. This work was supported by a grant from Ministry of Health and Welfare (HMP-00-B-20200-0012) to Jm.K. Jh.K. was supported by the BK21 program administered by the Ministry of Education, Republic of Korea (1999).

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