A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance

Souza, Cleiton Martins; Schwabe, Tatjana M.E.; Pichler, Harald; Ploier, Birgit; Leitner, Erich; Guan, Xue Li; Wenk, Markus R.; Riezman, Isabelle; Riezman, Howard

doi:10.1016/j.ymben.2011.06.006

Cited by 103 publications

(132 citation statements)

References 63 publications

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“…30 An overview of the cholesterol biosynthetic pathway and principles of biosynthetic isotopic labeling has been published. 31 Yeast growth media for isotopic labeling were 0.7% yeast nitrogen base (US Biological), 0.5% yeast extract (BD), 1.25% glucose, and 30 mg∕L uracil and leucine in deuterium oxide (99.8%, ARMAR, Switzerland).…”

Section: D38-cholesterolmentioning

confidence: 99%

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D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage

et al. 2015

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Abstract. We generated a highly deuterated cholesterol analog (D38-cholesterol) and demonstrated its use for selective vibrational imaging of cholesterol storage in mammalian cells. D38-cholesterol produces detectable signals in stimulated Raman scattering (SRS) imaging, is rapidly taken up by cells, and is efficiently metabolized by acyl-CoA cholesterol acyltransferase to form cholesteryl esters. Using hyperspectral SRS imaging of D38-cholesterol, we visualized cholesterol storage in lipid droplets. We found that some lipid droplets accumulated preferentially unesterified D38-cholesterol, whereas others stored D38-cholesteryl esters. In steroidogenic cells, D38-cholesteryl esters and triacylglycerols were partitioned into distinct sets of lipid droplets. Thus, hyperspectral SRS imaging of D38-cholesterol demonstrates a heterogeneous incorporation of neutral lipid species, i.e., free cholesterol, cholesteryl esters, and triacylglycerols, between individual lipid droplets in a cell.

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Section: D38-cholesterolmentioning

confidence: 99%

“…The deuterated cholesterol was purified from harvested cells and analyzed by gas chromatography-mass spectrometry (GC-MS) as described. 30 The yield was approximately 10 mg purified deuterated cholesterol per liter of cell culture. Analysis of the GC-MS profile of the deuterated cholesterol showed a GC profile [ Fig.…”

Section: D38-cholesterolmentioning

confidence: 99%

D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage

et al. 2015

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“…This required manipulation of activities other than CYPs and the yeast pathway was diverted by expression of a plant and eight mammalian proteins, including CYP11A1, CYP11B1, CYP17A1 and CYP21A1. A recent paper also discusses the alteration of yeast to a form producing cholesterol efficiently instead of ergosterol [108]. Other ambitious examples of pathway engineering that required CYP as an important player are the production of the anti-malarial drug precursor artemisinic acid using yeast [109] and taxol precursor in E. coli [110].…”

Section: Industrial Biotechnology and A Sustainable Futurementioning

confidence: 99%

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

Kelly

2013

Phil. Trans. R. Soc. B

177

131

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The first eukaryote genome revealed three yeast cytochromes P450 (CYPs), hence the subsequent realization that some microbial fungal genomes encode these proteins in 1 per cent or more of all genes (greater than 100) has been surprising. They are unique biocatalysts undertaking a wide array of stereo-and regio-specific reactions and so hold promise in many applications. Based on ancestral activities that included 14a-demethylation during sterol biosynthesis, it is now seen that CYPs are part of the genes and metabolism of most eukaryotes. In contrast, Archaea and Eubacteria often do not contain CYPs, while those that do are frequently interesting as producers of natural products undertaking their oxidative tailoring. Apart from roles in primary and secondary metabolism, microbial CYPs are actual/potential targets of drugs/agrochemicals and CYP51 in sterol biosynthesis is exhibiting evolution to resistance in the clinic and the field. Other CYP applications include the first industrial biotransformation for corticosteroid production in the 1950s, the diversion into penicillin synthesis in early mutations in fungal strain improvement and bioremediation using bacteria and fungi. The vast untapped resource of orphan CYPs in numerous genomes is being probed and new methods for discovering function and for discovering desired activities are being investigated.

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“…Membranes that incorporate ergosterol are more ordered and therefore thicker and less fluid than those that incorporate the same concentration of cholesterol 58 and could therefore account for this difference in stability of the tail anchor's organelle-specific insertion in yeast versus mammalian cells. To test this, we used a recently reported yeast strain in which intracellular ergosterol is replaced by cholesterol through exchange of the two terminal enzymes in the ergosterol production pathway with their cholesterol-specific counterparts 59 . Localization of the tail anchor of PTP1B in this mutant strain (cholesterol) was identical to that in its wild-type (ergosterol) counterpart (Fig.…”

Section: Investigation Of Tail Anchor Targeting Pathways That May Regmentioning

confidence: 99%

“…Yeast strains. All strains with an AK prefix -and therefore not ESM356-1 91 , RH2881 59 , and RH6829 59 -were generated for this study.…”

Section: Rh2881 (Wt)mentioning

confidence: 99%

Journey to the Center of the Mitochondria Guided by the Tail Anchor of Protein Tyrosine Phosphatase 1B

Fueller

Egorov

et al. 2013

Preprint

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The canonical protein tyrosine phosphatase PTP1B has traditionally been considered to exclusively reside on the endoplasmic reticulum (ER). Using confocal microscopy, we show that endogenous PTP1B actually exhibits a higher local concentration at the mitochondria in all mammalian cell lines that we tested. Fluorescently labeled chimeras containing full-length PTP1B or only its 35 amino acid tail anchor localized identically, demonstrating the complete dependence of PTP1B's subcellular partitioning on its tail anchor. Correlative light and electron microscopy using GFPdriven photo-oxidation of DAB revealed that PTP1B's tail anchor localizes it to the mitochondrial interior and to mitochondrial-associated membrane (MAM) sites along the ER. Heterologous expression of the tail anchor of PTP1B in the yeast S. cerevisiae surprisingly led to its exclusive localization to the ER/vacuole with no presence at the mitochondria. Studies with various yeast mutants of conserved membrane insertion pathways revealed a role for the GET/TRC40 pathway in ER insertion, but also emphasized the likely dominant role of spontaneous insertion. Further studies of modified tail isoforms in both yeast and mammalian cells revealed a remarkable sensitivity of subcellular partitioning to slight changes in transmembrane domain (TMD) length, C-terminal charge, and hydropathy. For example, addition of a single positive charge to the tail anchor was sufficient to completely shift the tail anchor to the mitochondria in mammalian cells and to largely shift it there in yeast cells, and a point mutation that increased TMD hydropathy was sufficient to localize the tail anchor exclusively to the ER in mammalian cells. Striking differences in the subcellular partitioning of a given tail anchor isoform in mammalian versus yeast cells most likely point to fundamental differences in the lipid composition of specific organelles (e.g. affecting membrane charge or thickness) in higher versus lower eukaryotes. Fluorescence lifetime imaging microscopy (FLIM) detection of the Förster Resonance Energy Transfer (FRET)-based interaction of the catalytic domain of PTP1B with the epidermal growth factor receptor (EGFR/ErbB1) at the mitochondria revealed a strong interaction on the cytosolic face of the outer mitochondrial membrane (OMM), suggesting the presence of a significant pool of PTP1B there and a novel role for PTP1B in the regulation of mitochondrial ErbB1 activity. In summary, in addition to its well-established general localization along the ER, our results reveal that PTP1B specifically accumulates at MAM sites along the ER and localizes as well to the OMM and mitochondrial matrix. Further elucidation of PTP1B's roles in these different locations (including the identification of its targets) will likely be critical for understanding its complex regulation of general cellular responses, cell proliferation, and diseased states.

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A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance

Cited by 103 publications

References 63 publications

D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage

D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

Journey to the Center of the Mitochondria Guided by the Tail Anchor of Protein Tyrosine Phosphatase 1B

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