Yasmine M. Mamnun

The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement

In the present study, we investigated a new member of the ABC transporter superfamily of Arabidopsis thaliana, AtMRP5. AtMRP5 encodes a 167 kDa protein and exhibits low glutathione conjugate and glucuronide conjugate transport activity. Promotor‐β‐glucuronidase fusion constructs showed that AtMRP5 is expressed mainly in the vascular bundle and in the epidermis, especially guard cells. Using reverse genetics, we identified a plant with a T‐DNA insertion in AtMRP5 (mrp5‐1). mrp5‐1 exhibited decreased root growth and increased lateral root formation. Auxin levels in the roots of mrp5‐1 plants were increased. This observation may indicate that AtMRP5 works as an auxin conjugate transporter or that mutant plants are affected in ion uptake, which may lead to changes in auxin concentrations. Experiments on epidermal strips showed that in contrast to wild type, the sulfonylurea glibenclamide had no effect on stomatal opening in mrp5‐1 plants. This result strongly suggests that AtMRP5 may also function as an ion channel regulator.

War1p, a Novel Transcription Factor Controlling Weak Acid Stress Response in Yeast

ABSTRACT The Saccharomyces cerevisiae ATP-binding cassette (ABC) transporter Pdr12p effluxes weak acids such as sorbate and benzoate, thus mediating stress adaptation. In this study, we identify a novel transcription factor, War1p, as the regulator of this stress adaptation through transcriptional induction of PDR12. Cells lacking War1p are weak acid hypersensitive, since they fail to induce Pdr12p. The nuclear Zn2Cys6 transcriptional regulator War1p forms homodimers and is rapidly phosphorylated upon sorbate stress. The appearance of phosphorylated War1p isoforms coincides with transcriptional activation of PDR12. Promoter deletion analysis identified a novel cis-acting weak acid response element (WARE) in the PDR12 promoter required for PDR12 induction. War1p recognizes and decorates the WARE both in vitro and in vivo, as demonstrated by band shift assays and in vivo footprinting. Importantly, War1p occupies the WARE in the presence and absence of stress, demonstrating constitutive DNA binding in vivo. Our results suggest that weak acid stress triggers phosphorylation and perhaps activation of War1p. In turn, War1p activation is necessary for the induction of PDR12 through a novel signal transduction event that elicits weak organic acid stress adaptation.

Fungal ABC proteins: pleiotropic drug resistance, stress response and cellular detoxification.

A number of prominent genetic diseases are caused by mutations in genes encoding ATP-binding cassette (ABC) proteins (Ambudkar, Gottesmann, 1998). Moreover, several mammalian ABC proteins such as P-glycoprotein (P-gp) (Gottesman et al., 1995) and multidrug-resistance-associated proteins (MRPs) (Cole, Deeley, 1998) have been implicated in multidrug resistance (MDR) phenotypes of tumor cells highly resistant to many different anticancer drugs. The characteristics of MDR phenomena include the initial resistance to a single anticancer drug, followed by the development of cross-resistance to many structurally and functionally unrelated drugs. Similar mechanisms of MDR exist in pathogenic fungi, including Candida and Aspergillus (Vanden Bossche et al., 1998), and also in parasites such as Plasmodium and Leishmania (Ambudkar, Gottesmann, 1998), as well as in many bacterial pathogens (Nikaido, 1998). To dissect the mechanisms of MDR development and to elucidate the physiological functions of ABC proteins, many efforts have been made during the past decade. Importantly, yeast orthologues of mammalian disease genes made this unicellular eukaryote an invaluable model system for studies on the molecular mechanisms of ABC proteins, in order to better understand and perhaps improve treatment of ABC gene-related disease. In this review, we provide an overview of ABC proteins and pleiotropic drug resistance in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. Furthermore, we discuss the role of ABC proteins in clinical drug resistance development of certain fungal pathogens.

The yeast zinc finger regulators Pdr1p and Pdr3p control pleiotropic drug resistance (PDR) as homo‐ and heterodimers in vivo

The transcription factors Pdr1p and Pdr3p from Saccharomyces cerevisiae mediate pleiotropic drug resistance (PDR) by controlling expression of ATP‐binding cassette (ABC) transporters such as Pdr5p, Snq2p and Yor1p. Previous in vitro studies demonstrated that Pdr1p and Pdr3p recognize so‐called pleiotropic drug resistance elements (PDREs) in the promoters of target genes. In this study, we show that both Pdr1p and Pdr3p are phosphoproteins; Pdr3p isoforms migrate as two bands in gel electrophoresis, reflecting two distinct phosphorylation states. Most importantly, native co‐immunoprecipitation experiments, using functional epitope‐tagged Pdr1p/Pdr3p variants, demonstrate that Pdr1p and Pdr3p can form both homo‐ and heterodimers in vivo. Furthermore, in vivo footprinting of PDRE‐containing promoters demonstrate that Pdr1p/Pdr3p constitutively occupy both perfect and degenerate PDREs in vivo. Thus, in addition to interaction with other regulators, differential dimerization provides a plausible explanation for the observation that Pdr3p and Pdr1p can both positively and negatively control PDR promoters with different combinations of perfect and degenerate PDREs.

The ATP-binding cassette (ABC) transporter Bpt1p mediates vacuolar sequestration of glutathione conjugates in yeast

Vacuolar sequestration or cellular extrusion of glutathione‐conjugated xenobiotics and catabolites by ATP‐binding cassette (ABC) transporters is an important detoxification mechanism operating in many species. In this study, we show that the yeast ABC transporter Bpt1p, a paralogue of Ycf1p, acts as an ATP‐dependent vacuolar pump for glutathione conjugates. Bpt1p, which is inhibited by vanadate and glibenclamide, accounts for one third of the total vacuolar transport of glutathione conjugates. Furthermore, immunoblot analyses show that Bpt1p levels are strongly elevated in early stationary phase, consistent with a function of Bpt1p in vacuolar detoxification.

Expression regulation of the yeast PDR5 ATP-binding cassette (ABC) transporter suggests a role in cellular detoxification during the exponential growth phase.

The yeast ATP‐binding cassette transporter Pdr5p mediates pleiotropic drug resistance (PDR) by effluxing a variety of xenobiotics. Immunoblotting demonstrates that Pdr5p levels are high in the logarithmic growth phase, while its levels decrease sharply when cells exit exponential growth. Here, we show that PDR5 promoter activity is dramatically reduced when cells stop growing due to a limitation of glucose or nitrogen or when they approach stationary phase. Interestingly, Pdr3p, a major transcriptional regulator of PDR5, shows the same regulatory pattern. Feeding glucose to starved cells rapidly re‐induces both PDR5 and PDR3 transcription. Importantly, diminished Pdr5p levels, as present after starvation, are rapidly restored in response to xenobiotic challenges that activate the transcription factors Pdr1p and Pdr3p. Our data indicate a role for yeast Pdr5p in cellular detoxification during exponential growth.

A new thermosensitive smc-3 allele reveals involvement of cohesin in homologous recombination in C. elegans.

The cohesin complex is required for the cohesion of sister chromatids and for correct segregation during mitosis and meiosis. Crossover recombination, together with cohesion, is essential for the disjunction of homologous chromosomes during the first meiotic division. Cohesin has been implicated in facilitating recombinational repair of DNA lesions via the sister chromatid. Here, we made use of a new temperature-sensitive mutation in the Caenorhabditis elegans SMC-3 protein to study the role of cohesin in the repair of DNA double-strand breaks (DSBs) and hence in meiotic crossing over. We report that attenuation of cohesin was associated with extensive SPO-11–dependent chromosome fragmentation, which is representative of unrepaired DSBs. We also found that attenuated cohesin likely increased the number of DSBs and eliminated the need of MRE-11 and RAD-50 for DSB formation in C. elegans, which suggests a role for the MRN complex in making cohesin-loaded chromatin susceptible to meiotic DSBs. Notably, in spite of largely intact sister chromatid cohesion, backup DSB repair via the sister chromatid was mostly impaired. We also found that weakened cohesins affected mitotic repair of DSBs by homologous recombination, whereas NHEJ repair was not affected. Our data suggest that recombinational DNA repair makes higher demands on cohesins than does chromosome segregation.

Mutations in Caenorhabditis elegans him-19 show meiotic defects that worsen with age.

Faithful meiotic chromosome segregation requires pairing, synapsis and recombination of homologous chromosomes. In mammals, chromosomal non-disjunction increases with age. A mutation in Caenorhabditis elegans him-19 mimics these age-dependent chromosome segregation defects and might therefore further our understanding of this phenomenon.