Functional roles of YPT31 and YPT32 in clotrimazole resistance of Saccharomyces cerevisiae through effects on vacuoles and ATP-binding cassette transporter(s)

Tsujimoto, Yasuhiro; Takase, Daisuke; Okano, Hajime; Tomari, Naohiro; Watanabe, Kunihiko; Matsui, Hiroshi

doi:10.1016/j.jbiosc.2012.08.011

Cited by 5 publications

(11 citation statements)

References 50 publications

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“…To construct genomic library, genomic DNA of S288C was partially digested with Sau3AI, and the resulting DNA fragments (approximately 3-8 kb) were collected and cloned into the Bam HI site of YEp24 multicopy vector (New England Biolabs, MA, USA) as described before. 16) Yeast strain YPH250 was used as a host, and clones which grew on SC medium containing 7 mM caffeine were selected and used in further analysis.…”

Section: Methodsmentioning

confidence: 99%

“…DNA fragments (snq2Δ::kanMX4) for SNQ2 knockout were prepared from the genomic DNA of the ID 3951 (snq2Δ:: kanMX4) as described. 16) Knockout of SNQ2 was performed in the YPH250 (wild type) and DY5c (pdr5Δ:: CgHIS3) cells by the one-step gene-replacement method to obtain DS2 (snq2Δ::kanMX4) and KY25 (snq2Δ::kanMX4 pdr5Δ::CgHIS3). Deletion of SNQ2 by the gene knockout was verified by PCR.…”

Section: Methodsmentioning

confidence: 99%

“…Assay of R6G-efflux activity. The R6G-efflux activity was measured according to the modified method of Tsujimoto et al 16) In brief, cells were cultured as described above (TLC determination of caffeine content), harvested, and washed with 50 mM MES buffer (pH 6.0). The cells were suspended in R6G solution [50 mM of MES (pH 6.0), 100 μM R6G, 5 mM 2-deoxyglucose, and 10 mM sodium azide] and incubated for 30 min at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…Pdr5p is one of the best characterized ABC transporters and is known to be involved in resistance against various chemicals such as R6G and CTZ, 16,[22][23][24] although involvement of PDR5 to caffeine resistance has not been reported. Multicopy of PDR5 also conferred resistance to 10 mM caffeine, but its resistance was smaller than that of SNQ2 ( Fig.…”

Section: Snq2 and Pdr5 Mediate Caffeine Resistancementioning

confidence: 99%

“…11,12) CTZ is an azole antifungal agent and is exported by Pdr5p using an ATP energy. 16,23) R6G is a derivative of xanthene which is widely used as a substrate for transporter assay, and is also exported by Pdr5p in an energy-dependent manner. 16,[22][23][24] Multicopy of SNQ2 yielded significant resistance to R6G and 4-NQO, but not to CTZ (Fig.…”

Section: Substrate Specificity Of Snq2p and Pdr5pmentioning

confidence: 99%

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Multidrug resistance transporters Snq2p and Pdr5p mediate caffeine efflux in Saccharomyces cerevisiae

Tsujimoto

Shimizu

Otake

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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SNQ2 was identified as a caffeine-resistance gene by screening a genomic library of Saccharomyces cerevisiae in a multicopy vector YEp24. SNQ2 encodes an ATP-binding cassette transporter and is highly homologous to PDR5. Multicopy of PDR5 also conferred resistance to caffeine, while its resistance was smaller than that of SNQ2. Residual caffeine contents were analyzed after transiently exposing cells to caffeine. The ratios of caffeine contents were 21.3 ± 8.8% (YEp24-SNQ2) and 81.9 ± 8.7% (YEp24-PDR5) relative to control (YEp24, 100%). In addition, multicopies of SNQ2 or PDR5 conferred resistance to rhodamine 6G (R6G), which was widely used as a substrate for transport assay. R6G was exported by both transporters, and their efflux activities were inhibited by caffeine with half-maximal inhibitory concentrations of 5.3 ± 1.9 (YEp24-SNQ2) and 17.2 ± 9.6 mM (YEp24-PDR5). These results demonstrate that Snq2p is a more functional transporter of caffeine than Pdr5p in yeast cells.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Snq2 and Pdr5 Mediate Caffeine Resistancementioning

confidence: 99%

Section: Substrate Specificity Of Snq2p and Pdr5pmentioning

confidence: 99%

See 3 more Smart Citations

Multidrug resistance transporters Snq2p and Pdr5p mediate caffeine efflux in Saccharomyces cerevisiae

Tsujimoto

Shimizu

Otake

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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Identifying novel protein phenotype annotations by hybridizing protein–protein interactions and protein sequence similarities

et al. 2016

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Studies of protein phenotypes represent a central challenge of modern genetics in the post-genome era because effective and accurate investigation of protein phenotypes is one of the most critical procedures to identify functional biological processes in microscale, which involves the analysis of multifactorial traits and has greatly contributed to the development of modern biology in the post genome era. Therefore, we have developed a novel computational method that identifies novel proteins associated with certain phenotypes in yeast based on the protein-protein interaction network. Unlike some existing network-based computational methods that identify the phenotype of a query protein based on its direct neighbors in the local network, the proposed method identifies novel candidate proteins for a certain phenotype by considering all annotated proteins with this phenotype on the global network using a shortest path (SP) algorithm. The identified proteins are further filtered using both a permutation test and their interactions and sequence similarities to annotated proteins. We compared our method with another widely used method called random walk with restart (RWR). The biological functions of proteins for each phenotype identified by our SP method and the RWR method were analyzed and compared. The results confirmed a large proportion of our novel protein phenotype annotation, and the RWR method showed a higher false positive rate than the SP method. Our method is equally effective for the prediction of proteins involving in all the eleven clustered yeast phenotypes with a quite low false positive rate. Considering the universality and generalizability of our supporting materials and computing strategies, our method can further be applied to study other organisms and the new functions we predicted can provide pertinent instructions for the further experimental verifications.

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A novel chloroplast localized Rab GTPase protein CPRabA5e is involved in stress, development, thylakoid biogenesis and vesicle transport in Arabidopsis

et al. 2013

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A novel Rab GTPase protein in Arabidopsis thaliana, CPRabA5e (CP = chloroplast localized) is located in chloroplasts and has a role in transport. Transient expression of CPRabA5e:EGFP fusion protein in tobacco (Nicotiana tabacum) leaves, and immunoblotting using Arabidopsis showed localization of CPRabA5e in chloroplasts (stroma and thylakoids). Ypt31/32 in the yeast Saccharomyces cerevisiae are involved in regulating vesicle transport, and CPRabA5e a close homolog of Ypt31/32, restores the growth of the ypt31Δ ypt32(ts) mutant at 37 °C in yeast complementation. Knockout mutants of CPRabA5e displayed delayed seed germination and growth arrest during oxidative stress. Ultrastructural studies revealed that after preincubation at 4 °C mutant chloroplasts contained larger plastoglobules, lower grana, and more vesicles close to the envelopes compared to wild type, and vesicle formation being enhanced under oxidative stress. This indicated altered thylakoid development and organization of the mutants. A yeast-two-hybrid screen with CPRabA5e as bait revealed 13 interacting partner proteins, mainly located in thylakoids and plastoglobules. These proteins are known or predicted to be involved in development, stress responses, and photosynthesis related processes, consistent with the stress phenotypes observed. The results observed suggest a role of CPRabA5e in transport to and from thylakoids, similar to cytosolic Rab proteins involved in vesicle transport.

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Functional roles of YPT31 and YPT32 in clotrimazole resistance of Saccharomyces cerevisiae through effects on vacuoles and ATP-binding cassette transporter(s)

Cited by 5 publications

References 50 publications

Multidrug resistance transporters Snq2p and Pdr5p mediate caffeine efflux in Saccharomyces cerevisiae

Multidrug resistance transporters Snq2p and Pdr5p mediate caffeine efflux in Saccharomyces cerevisiae

Identifying novel protein phenotype annotations by hybridizing protein–protein interactions and protein sequence similarities

A novel chloroplast localized Rab GTPase protein CPRabA5e is involved in stress, development, thylakoid biogenesis and vesicle transport in Arabidopsis

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