Abstract
The fight against multidrug-resistant pathogens requires an understanding of the underlying cellular mechanisms. In this work, we isolate and characterize one of the multidrug resistance ...determinants in Kluyveromyces lactis, the KlPDR16 gene. We show that KlPdr16p (345 aa), which belongs to the KlPdr1p regulon, is a functional homologue of the Saccharomyces cerevisiae Pdr16p. Deletion of KlPDR16 resulted in hypersensitivity of K. lactis cells to antifungal azoles, oligomycin, rhodamine 6G, 4-nitroquinoline-N-oxide and alkali metal cations. The Klpdr16∆ mutation led to a decreased content of ergosterol in whole-cell extract. In spite of the hypersensitivity of Klpdr16∆ mutant cells to rhodamine 6G and oligomycin, the transcript level of the KlPDR5 gene and the rhodamine 6G efflux in the mutant was the same as in the parental strain. Increased accumulation of rhodamine 6G in Klpdr16∆ cells indicates that KlPDR16 limits the rate of passive drug diffusion across the membrane, without affecting the glucose-induced drug export. The results obtained show that KlPDR16, similar to its orthologues in other yeast species, influences the passive drug diffusion into the yeast cell.
KlPDR16 is a new multidrug resistance determinant in Kluyveromyces lactis
Transcription of yeast phospholipid biosynthesis structural genes, which contain an inositol-sensitive upstream activating sequence in their promoters, responds to the availability of the soluble ...precursors inositol and choline and to changes in phospholipid metabolism. The INO1 gene is deregulated (derepressed when inositol is present) under the conditions of increased phosphatidylcholine (PtdCho) turnover, as occurs in the sec14Delta cki1Delta strain (SEC14 encodes the major yeast phosphatidylinositol transfer protein; CKI1 encodes choline kinase of the cytidine diphosphate choline pathway of PtdCho biosynthesis). Five proteins (Sfhp) share sequence homology with phosphatidylinositol transfer protein Sec14p. Two (Sfh2p and Sfh4p), when overexpressed largely complement the otherwise essential Sec14p requirement concerning growth and secretion. In this study, we analysed the ability of Sec14 homologues to correct the defect in regulation of phospholipid biosynthesis resulting from defective or missing Sec14p. We also analysed how PtdCho turnover relates to the transcriptional regulation of phospholipid biosynthesis. The results show that (a) none of the Sec14 homologues was able to substitute for Sec14p in its regulatory aspects of phospholipid biosynthesis, (b) removal of phospholipase D activity corrected the aberrant INO1 gene regulation in yeast strains with otherwise high PtdCho turnover, and (c) increased steady-state phosphatidic acid levels correlated with derepressed levels of the INO1 gene. Overall, the results support the model in which high phosphatidic acid levels lead to derepression of the genes of phospholipid biosynthesis Henry, S.A. & Patton-Vogt, J.L. (1998) Prog. Nucleic Acid Res. Mol. Biol.61, 133-179.
Yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, Sec14p, is essential for protein transport from the Golgi apparatus and for the cell viability. It is instrumental in ...maintaining the lipid composition of the Golgi membranes to be compatible with vesicle biogenesis and the secretory process by coordination of PC and PI metabolism. To address the question to which extent PC transfer ability of Sec14p is required for its essential in vivo function we generated a Sec14p mutant unable to transfer PC between membranes in the in vitro assay. Yeast cells with this modified Sec14p
D115G as a sole Sec14p were viable with improved secretory activity compared to
sec14 deficient strain. Thus, in vitro PC transfer ability of Sec14p is not required for its essential function(s) in living cells, however, yeast cells having PC transfer deficient Sec14p
D115G as a sole Sec14p display regulatory abnormalities, including increased phospholipase D mediated PC turnover.
Development of a daily rhythmicity in transcription of a gene encoding a rate-limiting enzyme of melatonin biosynthesis, the arylalkylamine-
N-acetyltransferase (AA-NAT) was studied by northern blot ...analysis in pineal glands of 16 and 19-day-old embryos and 1, 4, 8, 11, and 14-day-old chicks. In a parallel experiment, melatonin content in pineal glands and plasma was measured. A significant rhythm of AA-NAT expression was found at embryonic day (ED) 16, the earliest day assayed in this experiment. Expression was low during the daytime and a clear signal was found in the middle of the darktime. The intensity of the signal was increasing during the ontogeny. The nocturnal pineal melatonin concentrations were increasing over the studied period (from ED 19 until post-embryonic day 21). Midnight plasma melatonin concentrations increased from ED19 to PD 3 and oscillated around this value afterwards. Data show that rhythmic expression of AA-NAT mRNA starts very early in development of chicken and plays a major role in melatonin rhythm generation during embryonic development.
Griac reports that the major mitochondrial PS decarboxylase gene of the yeast Saccharomyces cervisiae is transcriptionally regulated by inositol in a manner similar to that reported for other ...coregulated phospholipid biosynthetic genes.
Sterol esterification in Saccharomyces cerevisiae is catalyzed by two acyl-CoA:sterol acyltransferases encoded by the genes ARE1 and ARE2. Using double mutants in the HEM1 gene and individual ARE ...genes we demonstrated that the relative contribution of these two enzymes to sterol esterification was dependent on cellular heme status. Observed changes in sterol esterification could be explained by a different effect of heme on the transcription of both genes: while the ARE1 transcript level was elevated in heme-deficient and anaerobic cells, the ARE2 gene transcript was more abundant in aerobic cells competent for heme synthesis. Our results indicate that transcriptional regulation of ARE genes by heme and specific substrate preferences of Are1p and Are2p may be involved in the adaptation of yeast sterol metabolism to hypoxia.
In yeast, mutations in the CDP-choline pathway for phosphatidylcholine biosynthesis permit the cell to grow even when the SEC14 gene is completely deleted (Cleves, A., McGee, T., Whitters, E., ...Champion, K., Aitken, J., Dowhan, W., Goebl, M., and Bankaitis, V. (1991) Cell 64, 789–800). We report that strains carrying mutations in the CDP-choline pathway, such ascki1, exhibit a choline excretion phenotype due to production of choline during normal turnover of phosphatidylcholine. Cells carrying cki1 in combination withsec14ts, a temperature-sensitive allele in the gene encoding the phosphatidylinositol/phosphatidylcholine transporter, have a dramatically increased choline excretion phenotype when grown at the sec14ts -restrictive temperature. We show that the increased choline excretion in sec14 ts cki1 cells is due to increased turnover of phosphatidylcholine via a mechanism consistent with phospholipase D-mediated turnover. We propose that the elevated rate of phosphatidylcholine turnover insec14 ts cki1 cells provides the metabolic condition that permits the secretory pathway to function when Sec14p is inactivated.
As phosphatidylcholine turnover increases in sec14 ts cki1 cells shifted to the restrictive temperature, theINO1 gene (encoding inositol-1-phosphate synthase) is also derepressed, leading to an inositol excretion phenotype (Opi−). Misregulation of the INO1 gene has been observed in many strains with altered phospholipid metabolism, and the relationship between phosphatidylcholine turnover and regulation ofINO1 and other co-regulated genes of phospholipid biosynthesis is discussed.
The INO1 gene of yeast is expressed in logarithmically growing, wild-type cells when inositol is absent from the medium. However, the INO1 gene is repressed when inositol is present during ...logarithmic growth and it is also repressed as cells enter stationary phase whether inositol is present or not. In this report, we demonstrate that transient nitrogen limitation also causes INO1 repression. The repression of INO1 in response to nitrogen limitation shares many features in common with repression in response to the presence of inositol. Specifically, the response to nitrogen limitation is dependent upon the presence of a functional OPI1 gene product, it requires ongoing phosphatidylcholine biosynthesis and it is mediated by the repeated element, UASINO, found in the promoter of INO1 and other co-regulated genes of phospholipid biosynthesis. Thus, we propose that repression of INO1 in response to inositol and in response to nitrogen limitation occurs via a common mechanism that is sensitive to the status of ongoing phospholipid metabolism.
Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 ...(SCF(Grr1)). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway.
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