Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation

The protein kinase Gcn2 and its effector protein Gcn1 are part of the General Amino Acid Control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis.  Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P).  This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation.  Xrn1 is involved in RNA decay, quality control and processing.  We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex.  Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels.  Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion doesn't enhance eIF2α de-phosphorylation.  Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly.  Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.fals

A Plasmid System That Utilises Phosphoribosylanthranilate Isomerase to Select Against Cells Expressing Truncated Proteins

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Rapid yeast-based screen for Functionally Relevant Amino Acids (RS-FRAA) in a protein

Here, we describe a fast and cost-effective procedure to generate a large array of mutant proteins and immediately screen for those with altered protein function. This protocol is a modification from three existing approaches: fusion PCR, Saccharomyces cerevisiae in-yeast recombination, and semi-quantitative growth assays. We also describe a mating step to reduce the occurrence of false positive findings due to ectopic mutations. The only requirement is that the protein elicits a phenotype in Saccharomyces cerevisiae.fals

Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals-Illustrated with Four Actin Cytoskeleton Proteins

The budding yeast Saccharomyces cerevisiae has an actin cytoskeleton that comprises a set of protein components analogous to those found in the actin cytoskeletons of higher eukaryotes. Furthermore, the actin cytoskeletons of S. cerevisiae and of higher eukaryotes have some similar physiological roles. The genetic tractability of budding yeast and the availability of a stable haploid cell type facilitates the application of molecular genetic approaches to assign functions to the various actin cytoskeleton components. This has provided information that is in general complementary to that provided by studies of the equivalent proteins of higher eukaryotes and hence has enabled a more complete view of the role of these proteins. Several human functional homologues of yeast actin effectors are implicated in diseases. A better understanding of the molecular mechanisms underpinning the functions of these proteins is critical to develop improved therapeutic strategies. In this article we chose as examples four evolutionarily conserved proteins that associate with the actin cytoskeleton: 1) yeast Hof1p/mammalian PSTPIP1, 2) yeast Rvs167p/mammalian BIN1, 3) yeast eEF1A/eEF1A1 and eEF1A2 and 4) yeast Yih1p/mammalian IMPACT. We compare the knowledge on the functions of these actin cytoskeleton-associated proteins that has arisen from studies of their homologues in yeast with information that has been obtained from in vivo studies using live animals or in vitro studies using cultured animal cell lines.fals

GCN2 in Viral Defence and the Subversive Tactics Employed by Viruses

The recent SARS-CoV-2 pandemic and associated COVID19 disease illustrates the important role of viral defence mechanisms in ensuring survival and recovery of the host or patient. Viruses absolutely depend on the host's protein synthesis machinery to replicate, meaning that impeding translation is a powerful way to counteract viruses. One major approach used by cells to obstruct protein synthesis is to phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Mammals possess four different eIF2α-kinases: PKR, HRI, PEK/PERK, and GCN2. While PKR is currently considered the principal eIF2α-kinase involved in viral defence, the other eIF2α-kinases have also been found to play significant roles. Unsurprisingly, viruses have developed mechanisms to counteract the actions of eIF2α-kinases, or even to exploit them to their benefit. While some of these virulence factors are specific to one eIF2α-kinase, such as GCN2, others target all eIF2α-kinases. This review critically evaluates the current knowledge of viral mechanisms targeting the eIF2α-kinase GCN2. A detailed and in-depth understanding of the molecular mechanisms by which viruses evade host defence mechanisms will help to inform the development of powerful anti-viral measures.fals

Advancing urban transitions and transformations research

Urban transitions and transformations research fosters a dialogue between sustainability transitions theory an inter- and transdisciplinary research on urban change. As a field, urban transitions and transformations research encompasses plural analytical and conceptual perspectives. In doing so, this field opens up sustainability transitions research to new communities of practice in urban environments, including mayors, transnational municipal networks, and international organizations

Re-using Criterion plastic precast gel cassettes for SDS-polyacrylamide electrophoresis.

Precast gels are made with plastic cassettes which usually are discarded after use. Here we describe how Criterion plastic gel cassettes can be re-used for making SDS-PAGE gels in-house

Ghuge et al Sattlegger 2022 - Data S2

Data S2: Sample calculations, to determine the growth rate of strains in Fig. 7A.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV