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首页> 外文学位 >Intracellular sterol transport and distribution in Saccharomyces cerevisiae.
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Intracellular sterol transport and distribution in Saccharomyces cerevisiae.

机译：酿酒酵母中细胞内固醇的运输和分布。

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摘要

Sterols, such as cholesterol in mammals and ergosterol in yeast, are important components of cellular membranes. Sterol biosynthesis is tightly regulated because deviations from normal sterol levels have deleterious effects on numerous cellular processes. Although their biosynthesis has been extensively studied, relatively little is known about the mechanisms controlling the intracellular transport and distribution of sterols. Recent work indicates sterols move between intracellular membranes by non-vesicular mechanisms.;To identify components of the non-vesicular transport pathway, we used a tritium suicide selection to isolate yeast mutants defective in sterol transport. The selection is based on the transport-dependent incorporation of [ 3H]cholesterol and utilized the Upc2-1 strain of yeast, which is capable of incorporating exogenous sterols under aerobic conditions. Incorporated [3H]cholesterol is transported from the PM to the endoplasmic reticulum (ER) where it is esterified and stored in lipid droplets. When stored at -80°C, cells that efficiently incorporated the exogenous sterol suffered radiation-induced DNA damage and died. Using this selection we identified a number of genes that, when disrupted, significantly reduced the accumulation of exogenous sterols and allowed the cells to survive. One mutant, disrupted in DET1, was defective in sterol transport between the ER and the PM. Lipid analyses of det1Delta showed that this defect was not due to altered sterol levels or localization, suggesting that Det1 plays a critical role in intracellular sterol transport.;We also examined the transbilayer distribution of sterol in live yeast. The hem1Delta strain is auxotrophic for sterols and consequently requires exogenous sterols for growth. hem1Delta efficiently incorporated the fluorescent sterol dehydroergosterol (DHE). DHE functionally replaced ergosterol and assumed a similar intracellular distribution with ∼70% present at the PM. Membrane impermeant quenchers added extracellularly reduced DHE fluorescence by ∼20%. When the quencher was able to access internal membranes, DHE fluorescence was reduced by ∼75%. Disruption of cellular ATP levels or the PM electrochemical gradient across had no additional effect on fluorescence reduction. Together these data indicate that the majority of PM sterol is localized to the inner leaflet and is maintained there by a process independent of energy and the PM electrochemical gradient.
机译：甾醇，例如哺乳动物的胆固醇和酵母中的麦角甾醇，是细胞膜的重要成分。严格地控制甾醇的生物合成，因为偏离正常固醇水平会对许多细胞过程产生有害影响。尽管已经对其生物合成进行了广泛的研究，但是关于控制固醇的细胞内转运和分布的机理知之甚少。最近的工作表明固醇通过非囊泡机制在细胞内膜之间移动。为了确定非囊泡运输途径的组成，我们使用了tri自杀选择来分离固醇运输缺陷的酵母突变体。该选择基于[3 H]胆固醇的运输依赖性掺入，并利用了酵母的Upc2-1菌株，该菌株能够在有氧条件下掺入外源固醇。掺入的[3H]胆固醇从PM转运到内质网（ER），在那里被酯化并存储在脂质滴中。当储存在-80°C时，有效掺入外源固醇的细胞会遭受辐射诱导的DNA损伤并死亡。使用这种选择，我们鉴定了许多基因，这些基因被破坏后会显着减少外源固醇的积累，并使细胞存活。在DET1中被破坏的一个突变体在ER和PM之间的固醇转运存在缺陷。 det1Delta的脂质分析表明，该缺陷不是由于固醇水平或位置的改变所致，这表明Det1在细胞内固醇转运中起着关键作用。我们还研究了活酵母中固醇的双分子分布。 hem1Delta菌株对固醇是营养缺陷的，因此需要外源固醇才能生长。 hem1Delta有效地结合了荧光固醇脱氢麦角固醇（DHE）。 DHE在功能上取代了麦角固醇，并具有类似的细胞内分布，其中PM含量约为70％。膜不渗透猝灭剂添加了细胞外DHE荧光，降低了约20％。当淬灭剂能够进入内膜时，DHE荧光降低约75％。细胞ATP水平或PM电化学梯度的破坏对荧光降低没有其他影响。这些数据加在一起表明，大多数PM固醇位于内部小叶上，并通过独立于能量和PM电化学梯度的过程保持在那里。

著录项

  • 作者

    Sullivan, David Patrick.;

  • 作者单位

    Weill Medical College of Cornell University.;

  • 授予单位 Weill Medical College of Cornell University.;
  • 学科 Biology Cell.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 173 p.
  • 总页数 173
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;生物化学;
  • 关键词

  • 入库时间 2022-08-17 11:38:25

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