1887

Abstract

Sterols are a major class of membrane lipids in eukaryotes. In , sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of or did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in Δ mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in Δ but not in other mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the Δ mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in . Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins.

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2019-10-14
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Comparison of fission yeast (Sp) and budding yeast (Sc) Erg proteins. The black boxes indicate the conserved regions between Sp and Sc Erg proteins. The percentages of identical residues determined by BLAST are also indicated. [ PDF] (12 kb) HPLC separation of sterols from ARC039 (wild-type, WT), Δ and Δ single mutants. The dashed line indicates the retention time of ergosterol. [ PDF] (229 kb) Localization of ABC transporters in Δ cells. Plasmids pTN197/pmd1 and pTN197/bfr1 were used to express a GFP-tagged ABC transporter (Iwaki , 2006). (a) Localization of Pmd1-GFP. Cells carrying pTN197/pmd1 were cultured in minimal medium without leucine and thiamine for 20 h. Pmd1-GFP was found in the plasma membrane of Δ cells. (b) Localization of Bfr1-GFP. Cells carrying pTN197/bfr1 were cultured in minimal medium without leucine and thiamine for 20 h. Bfr1-GFP localized to the plasma membrane in Δ cells. [ PDF] (170 kb) A survey of all 11 ABC transporters in fission yeast: two novel ABC transporers are required for red pigment accumulation in a adenine biosynthetic mutant. , 2309-2321.

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Comparison of fission yeast (Sp) and budding yeast (Sc) Erg proteins. The black boxes indicate the conserved regions between Sp and Sc Erg proteins. The percentages of identical residues determined by BLAST are also indicated. [ PDF] (12 kb) HPLC separation of sterols from ARC039 (wild-type, WT), Δ and Δ single mutants. The dashed line indicates the retention time of ergosterol. [ PDF] (229 kb) Localization of ABC transporters in Δ cells. Plasmids pTN197/pmd1 and pTN197/bfr1 were used to express a GFP-tagged ABC transporter (Iwaki , 2006). (a) Localization of Pmd1-GFP. Cells carrying pTN197/pmd1 were cultured in minimal medium without leucine and thiamine for 20 h. Pmd1-GFP was found in the plasma membrane of Δ cells. (b) Localization of Bfr1-GFP. Cells carrying pTN197/bfr1 were cultured in minimal medium without leucine and thiamine for 20 h. Bfr1-GFP localized to the plasma membrane in Δ cells. [ PDF] (170 kb) A survey of all 11 ABC transporters in fission yeast: two novel ABC transporers are required for red pigment accumulation in a adenine biosynthetic mutant. , 2309-2321.

PDF

Comparison of fission yeast (Sp) and budding yeast (Sc) Erg proteins. The black boxes indicate the conserved regions between Sp and Sc Erg proteins. The percentages of identical residues determined by BLAST are also indicated. [ PDF] (12 kb) HPLC separation of sterols from ARC039 (wild-type, WT), Δ and Δ single mutants. The dashed line indicates the retention time of ergosterol. [ PDF] (229 kb) Localization of ABC transporters in Δ cells. Plasmids pTN197/pmd1 and pTN197/bfr1 were used to express a GFP-tagged ABC transporter (Iwaki , 2006). (a) Localization of Pmd1-GFP. Cells carrying pTN197/pmd1 were cultured in minimal medium without leucine and thiamine for 20 h. Pmd1-GFP was found in the plasma membrane of Δ cells. (b) Localization of Bfr1-GFP. Cells carrying pTN197/bfr1 were cultured in minimal medium without leucine and thiamine for 20 h. Bfr1-GFP localized to the plasma membrane in Δ cells. [ PDF] (170 kb) A survey of all 11 ABC transporters in fission yeast: two novel ABC transporers are required for red pigment accumulation in a adenine biosynthetic mutant. , 2309-2321.

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