Coenzyme Q (CoQ, ubiquinone) is an essential component of the electron transport system in aerobic organisms. Human type CoQ10, which has 10 units of isoprene in its quinone structure, is especially valuable as a food supplement. Therefore, studying the biosynthesis of CoQ10 is important not only for increasing metabolic knowledge, but also for improving biotechnological production. Herein, we show that Schizosaccharomyces pombe utilizes p-aminobenzoate (PABA) in addition to p-hydroxybenzoate (PHB) as a precursor for CoQ10 synthesis. We explored compounds that affect the synthesis of CoQ10 and found benzoic acid (Bz) at >5 μg/mL inhibited CoQ biosynthesis without accumulation of apparent CoQ intermediates. This inhibition was counteracted by incubation with a 10-fold lower amount of PABA or PHB. Overexpression of PHB-polyprenyl transferase encoded by ppt1 (coq2) also overcame the inhibition of CoQ biosynthesis by Bz. Inhibition by Bz was efficient in S. pombe and Schizosaccharomyces japonicus, but less so in Saccharomyces cerevisiae, Aureobasidium pullulans, and Escherichia coli. Bz also inhibited a S. pombe ppt1 (coq2) deletion strain expressing human COQ2, and this strain also utilized PABA as a precursor of CoQ10. Thus, Bz is likely to inhibit prenylation reactions involving PHB or PABA catalyzed by Coq2.
The amino acid proline functions as a nitrogen source and as a stress protectant
in the yeast Saccharomyces cerevisiae. However, utilization of
proline as a carbon source in S. cerevisiae cells has not been
studied yet. In the process of study on the physiological roles of the
found-in-mitochondrial-proteome (FMP) genes in proline
metabolism, we found that Δfmp12 cells could grow better than
wild-type cells on agar plate medium containing proline as the sole nitrogen and
carbon sources. In contrast, overexpression of FMP12 negatively
affected cell growth under the same condition. The Fmp12 protein was localized
in the mitochondria and was constitutively expressed. Deletion of the genes that
encode mitochondrial enzymes, such as proline dehydrogenase
(PUT1), Δ1-pyrroline-5-carboxylate dehydrogenase
(PUT2), alanine transaminase (ALT1), and
α-ketoglutarate dehydrogenase subunit (KGD1), abolished the
enhanced cell growth in Δfmp12. These results provided the
first evidence that proline can be utilized as a carbon source via the
mitochondrial proline metabolic pathway and the subsequent tricarboxylic acid
(TCA) cycle in S. cerevisiae. The function of Fmp12, which has
a similarity with α-ketoglutarate-dependent dioxygenases of the yeast
Candida species and human, might inhibit cell growth by
skipping the ATP production step of the TCA cycle.
We demonstrate a novel bio-spectroscopic technique, "simultaneous Raman/GFP microspectroscopy". It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti-Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two-component multivariate curve resolution with alternate least squares (MCR-ALS) analysis in the anti-Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR-ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm −1 , previously reported as "Raman spectroscopic signature of life ofanti-Stokes Raman spectroscopy, mitochondria, Raman spectroscopic signature of life, Schizosaccharomyces pombe, simultaneous Raman/GFP microspectroscopy, yeast
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