Localized inhibition of protein phosphatase 1 by NUAK1 promotes spliceosome activity and reveals a MYC-sensitive feedback control of transcription.

Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes

Cytosolic Fe-S cluster protein maturation and iron regulation are independent of the mitochondrial Erv1/Mia40 import system

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis

AglH, a thermophilic UDP‑<i>N</i>‑acetylglucosamine‑1‑phosphate:dolichyl phosphate GlcNAc‑1‑phosphotransferase initiating protein<i> N</i>‑glycosylation pathway in <i>Sulfolobus acidocaldarius</i>, is capable of complementing the eukaryal Alg7

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D(100)), IV (F(220)) and V (F(264)) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival

Expression of a malarial Hsp70 improves defects in chaperone-dependent activities in ssa1 mutant yeast

Plasmodium falciparum causes the most virulent form of malaria and encodes a large number of molecular chaperones. Because the parasite encounters radically different environments during its lifecycle, many members of this chaperone ensemble may be essential for P. falciparum survival. Therefore, Plasmodium chaperones represent novel therapeutic targets, but to establish the mechanism of action of any developed therapeutics, it is critical to ascertain the functions of these chaperones. To this end, we report the development of a yeast expression system for PfHsp70-1, a P. falciparum cytoplasmic chaperone. We found that PfHsp70-1 repairs mutant growth phenotypes in yeast strains lacking the two primary cytosolic Hsp70s, SSA1 and SSA2, and in strains harboring a temperature sensitive SSA1 allele. PfHsp70-1 also supported chaperone-dependent processes such as protein translocation and ER associated degradation, and ameliorated the toxic effects of oxidative stress. By introducing engineered forms of PfHsp70-1 into the mutant strains, we discovered that rescue requires PfHsp70-1 ATPase activity. Together, we conclude that yeast can be co-opted to rapidly uncover specific cellular activities mediated by malarial chaperones. © 2011 Bell et al

Preparation and Use of a Yeast shRNA Delivery System for Gene Silencing in Mosquito Larvae

The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the dengue and Zika virus vector Aedes aegypti and the primary African malaria vector Anopheles gambiae. RNA interference (RNAi) has facilitated gene silencing experiments in both of these disease vector mosquito species and could one day be applied as a new method of vector control. Here, we describe a procedure for the genetic engineering of Saccharomyces cerevisiae (baker’s yeast) that express short hairpin RNA (shRNA) corresponding to mosquito target genes of interest. Following cultivation, which facilitates inexpensive propagation of shRNA, the yeast is inactivated and prepared in a ready-to-use dry tablet formulation that is fed to mosquito larvae. Ingestion of the yeast tablets results in effective larval target gene silencing. This technically straightforward and affordable technique may be applicable to a wide variety of mosquito species and potentially to other arthropods that feed on yeast

Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis

Biofysische structuurchemi

Reconstitution of Mdm2-Dependent Post-Translational Modifications of p53 in Yeast

p53 mediates cell cycle arrest or apoptosis in response to DNA damage. Its activity is subject to a tight regulation involving a multitude of post-translational modifications. The plethora of functional protein interactions of p53 at present precludes a clear understanding of regulatory principles in the p53 signaling network. To circumvent this complexity, we studied here the minimal requirements for functionally relevant p53 post-translational modifications by expressing human p53 together with its best characterized modifier Mdm2 in budding yeast. We find that expression of the human p53-Mdm2 module in yeast is sufficient to faithfully recapitulate key aspects of p53 regulation in higher eukaryotes, such as Mdm2-dependent targeting of p53 for degradation, sumoylation at lysine 386 and further regulation of this process by p14ARF. Interestingly, sumoylation is necessary for the recruitment of p53-Mdm2 complexes to yeast nuclear bodies morphologically akin to human PML bodies. These results suggest a novel role for Mdm2 as well as for p53 sumoylation in the recruitment of p53 to nuclear bodies. The reductionist yeast model that was established and validated in this study will now allow to incrementally study simplified parts of the intricate p53 network, thus helping elucidate the core mechanisms of p53 regulation as well as test novel strategies to counteract p53 malfunctions

Homologous Recombination Is Stimulated by a Decrease in dUTPase in Arabidopsis

Deoxyuridine triphosphatase (dUTPase) enzyme is an essential enzyme that protects DNA against uracil incorporation. No organism can tolerate the absence of this activity. In this article, we show that dUTPase function is conserved between E. coli (Escherichia coli), yeast (Saccharomyces cerevisiae) and Arabidopsis (Arabidopsis thaliana) and that it is essential in Arabidopsis as in both micro-organisms. Using a RNA interference strategy, plant lines were generated with a diminished dUTPase activity as compared to the wild-type. These plants are sensitive to 5-fluoro-uracil. As an indication of DNA damage, inactivation of dUTPase results in the induction of AtRAD51 and AtPARP2, which are involved in DNA repair. Nevertheless, RNAi/DUT1 constructs are compatible with a rad51 mutation. Using a TUNEL assay, DNA damage was observed in the RNAi/DUT1 plants. Finally, plants carrying a homologous recombination (HR) exclusive substrate transformed with the RNAi/DUT1 construct exhibit a seven times increase in homologous recombination events. Increased HR was only detected in the plants that were the most sensitive to 5-fluoro-uracils, thus establishing a link between uracil incorporation in the genomic DNA and HR. Our results show for the first time that genetic instability provoked by the presence of uracils in the DNA is poorly tolerated and that this base misincorporation globally stimulates HR in plants