Identification of a hypothetical membrane protein interactor of ribosomal phosphoprotein P0

The ribosomal phosphoprotein P0 of the human malarial parasitePlasmodium falciparum (PfP0) has been identified as a protective surface protein. InDrosophila, P0 protein functions in the nucleus. The ribosomal function of P0 is mediated at the stalk of the large ribosomal subunit at the GTPase centre, where the elongation factor eEF2 binds. The multiple roles of the P0 protein presumably occur through interactions with other proteins. To identify such interacting protein domains, a yeast two-hybrid screen was carried out. Out of a set of sixty clones isolated, twelve clones that interacted strongly with both PfP0 and theSaccharomyces cerevisiae P0 (ScP0) protein were analysed. These belonged to three broad classes: namely (i) ribosomal proteins; (ii) proteins involved in nucleotide binding; and (iii) hypothetical integral membrane proteins. One of the strongest interactors (clone 67B) mapped to the gene YFL034W which codes for a hypothetical integral membrane protein, and is conserved amongst several eukaryotic organisms. The insert of clone 67B was expressed as a recombinant protein, and immunoprecipitaion (IP) reaction with anti-P0 antibodies pulled down this protein along with PfP0 as well as ScP0 protein. Using deletion constructions, the domain of ScP0, which interacted with clone 67B, was mapped to 60–148 amino acids. It is envisaged that the surface localization of P0 protein may be mediated through interactions with putative YFL034W-like proteins inP. falciparum     

Hypersensitivity of Escherichia coli Delta(uvrB-bio) mutants to 6-hydroxylaminopurine and other base analogs is due to a defect in molybdenum cofactor biosynthesis

We have shown previously that Escherichia coli and Salmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the uvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrA deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the moeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the uvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.     

Complex interplay among regulators of drug resistance genes in Saccharomyces cerevisiae

The Gal4p family of yeast zinc cluster proteins comprises regulators of multidrug resistance genes. For example, Pdr1p and Pdr3p bind as homo- or heterodimers to pleiotropic drug response elements (PDREs) found in promoters of target genes. Other zinc cluster activators of multidrug resistance genes include Stb5p and Yrr1p. To better understand the interplay among these activators, we have performed native co-immunoprecipitation experiments using strains expressing tagged zinc cluster proteins from their natural chromosomal locations. Interestingly, Stb5p is found predominantly as a Pdr1p heterodimer and shows little homodimerization. No interactions of Stb5p with Pdr3p or Yrr1p could be detected in our assays. In contrast to Stb5p, Yrr1p is only detected as a homodimer. Similar results were obtained using glutathione S-transferase pull-down assays. Importantly, the purified DNA binding domains of Stb5p and Pdr1p bound to a PDRE as heterodimers in vitro. These results suggest that the DNA binding domains of Pdr1p and Stb5p are sufficient for heterodimerization. Our data demonstrate a complex interplay among these activators and suggest that Pdr1p is a master drug regulator involved in recruiting other zinc cluster proteins to fine tune the regulation of multidrug resistance genes.