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Cell Lysis in S. pombe ura4 Mutants Is Suppressed by Loss of Functional Pub1, Which Regulates the Uracil Transporter Fur4.

Nishino K, Kushima M, Matsuo Y, Matsuo Y, Kawamukai M - PLoS ONE (2015)

Bottom Line: Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP.Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner.In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Japan.

ABSTRACT
Schizosaccharomyces pombe Δura4 cells lyse when grown on YPD medium. A S. pombe non-essential gene deletion library was screened to determine suppressors of the lysis phenotype. Deletion of the pub1 gene, which encoded E3 ubiquitin ligase, strongly suppressed cell lysis in Δura4 cells. The Δpub1 cells displayed high sensitivity to 5-fluorouracil, a toxic analog of uracil, and this sensitivity was suppressed by deletion of fur4, which encoded a uracil transporter. Fur4 localized primarily to the Golgi apparatus and vacuoles in wild-type cells, but localization was predominantly at the plasma membrane in Δpub1 cells. Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP. Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner. In addition, uracil starvation was critical for induction of cell lysis of Δura4 strains and uracil supplementation suppressed lysis. In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background.

No MeSH data available.


Related in: MedlinePlus

Localization of Fur4-GFP under nitrogen starvation conditions.(A) KNP74(ura4+), and KNP95 (Δura4) cells expressing Fur4-GFP were grown in YES liquid medium for 12 h. Cells were washed twice with sterile water and suspended in EMM, EMMU, EMM(-N), or EMMU(-N) media and incubated at 30°C for 1 h. Cells were observed using fluorescence microscopy. Bar: 10 μm.
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pone.0141796.g004: Localization of Fur4-GFP under nitrogen starvation conditions.(A) KNP74(ura4+), and KNP95 (Δura4) cells expressing Fur4-GFP were grown in YES liquid medium for 12 h. Cells were washed twice with sterile water and suspended in EMM, EMMU, EMM(-N), or EMMU(-N) media and incubated at 30°C for 1 h. Cells were observed using fluorescence microscopy. Bar: 10 μm.

Mentions: In S. cerevisiae, Fur4p localizes to the membrane upon uracil starvation. To determine whether such regulation occurred in S. pombe, Fur4 was examined in wild-type and Δura4 cells, but no apparent difference in localization was observed (Fig 4). Localization of Fur4-GFP was no significant different when cells were cultured in long time (0, 6 or 12h) (S2 Fig). We next examined the localization of Fur4-GFP cells cultured in EMM, EMM(-N), EMMU, and EMMU(-N) medium. In contrast to a slight membrane localization of Fur4-GFP in cells grown in EMM and YES, clear membrane localization of it was observed in both wild-type and Δura4 cells grown in EMMU(-N) medium (Fig 4). Some membrane localization was also seen in EMM(-N) medium. This demonstrated that nitrogen starvation, but not uracil starvation, induced membrane localization of Fur4 in S. pombe, indicating that the regulation of Fur4 in S. pombe differed from that of Fur4p in S. cerevisiae.


Cell Lysis in S. pombe ura4 Mutants Is Suppressed by Loss of Functional Pub1, Which Regulates the Uracil Transporter Fur4.

Nishino K, Kushima M, Matsuo Y, Matsuo Y, Kawamukai M - PLoS ONE (2015)

Localization of Fur4-GFP under nitrogen starvation conditions.(A) KNP74(ura4+), and KNP95 (Δura4) cells expressing Fur4-GFP were grown in YES liquid medium for 12 h. Cells were washed twice with sterile water and suspended in EMM, EMMU, EMM(-N), or EMMU(-N) media and incubated at 30°C for 1 h. Cells were observed using fluorescence microscopy. Bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4633276&req=5

pone.0141796.g004: Localization of Fur4-GFP under nitrogen starvation conditions.(A) KNP74(ura4+), and KNP95 (Δura4) cells expressing Fur4-GFP were grown in YES liquid medium for 12 h. Cells were washed twice with sterile water and suspended in EMM, EMMU, EMM(-N), or EMMU(-N) media and incubated at 30°C for 1 h. Cells were observed using fluorescence microscopy. Bar: 10 μm.
Mentions: In S. cerevisiae, Fur4p localizes to the membrane upon uracil starvation. To determine whether such regulation occurred in S. pombe, Fur4 was examined in wild-type and Δura4 cells, but no apparent difference in localization was observed (Fig 4). Localization of Fur4-GFP was no significant different when cells were cultured in long time (0, 6 or 12h) (S2 Fig). We next examined the localization of Fur4-GFP cells cultured in EMM, EMM(-N), EMMU, and EMMU(-N) medium. In contrast to a slight membrane localization of Fur4-GFP in cells grown in EMM and YES, clear membrane localization of it was observed in both wild-type and Δura4 cells grown in EMMU(-N) medium (Fig 4). Some membrane localization was also seen in EMM(-N) medium. This demonstrated that nitrogen starvation, but not uracil starvation, induced membrane localization of Fur4 in S. pombe, indicating that the regulation of Fur4 in S. pombe differed from that of Fur4p in S. cerevisiae.

Bottom Line: Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP.Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner.In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Japan.

ABSTRACT
Schizosaccharomyces pombe Δura4 cells lyse when grown on YPD medium. A S. pombe non-essential gene deletion library was screened to determine suppressors of the lysis phenotype. Deletion of the pub1 gene, which encoded E3 ubiquitin ligase, strongly suppressed cell lysis in Δura4 cells. The Δpub1 cells displayed high sensitivity to 5-fluorouracil, a toxic analog of uracil, and this sensitivity was suppressed by deletion of fur4, which encoded a uracil transporter. Fur4 localized primarily to the Golgi apparatus and vacuoles in wild-type cells, but localization was predominantly at the plasma membrane in Δpub1 cells. Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP. Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner. In addition, uracil starvation was critical for induction of cell lysis of Δura4 strains and uracil supplementation suppressed lysis. In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background.

No MeSH data available.


Related in: MedlinePlus