GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2α in Arabidopsis

The yeast regulatory protein kinase, general control non-derepressible-2 (GCN2) plays a key role in general amino acid control. GCN2 phosphorylates the α subunit of the trimeric eukaryotic translation initiation factor-2 (eIF2), bringing about a decrease in the general rate of protein synthesis but an increase in the synthesis of GCN4, a transcription factor that promotes the expression of genes encoding enzymes for amino acid biosynthesis. The present study concerned the phosphorylation of Arabidopsis eIF2α (AteIF2α) by the Arabidopsis homologue of GCN2, AtGCN2, and the role of AtGCN2 in regulating genes encoding enzymes of amino acid biosynthesis and responding to virus infection. A null mutant for AtGCN2 called GT8359 was obtained and western analysis confirmed that it lacked AtGCN2 protein. GT8359 was more sensitive than wild-type Arabidopsis to herbicides that affect amino acid biosynthesis. Phosphorylation of AteIF2α occurred in response to herbicide treatment but only in wild-type Arabidopsis, not GT8359, showing it to be AtGCN2-dependent. Expression analysis of genes encoding key enzymes for amino acid biosynthesis and nitrate assimilation revealed little effect of loss of AtGCN2 function in GT8359 except that expression of a nitrate reductase gene, NIA1, was decreased. Analysis of wild-type and GT8359 plants infected with Turnip yellow mosaic virus or Turnip crinkle virus showed that AteIF2α was not phosphorylated

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

A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation

The General Amino Acid Control stress signalling pathway allows cells to sense and overcome starvation. One of the major players in this pathway is the protein kinase Gcn2 found in all eukaryotic cells. Activation of Gcn2 leads to phosphorylation of the alpha subunit of eukaryotic translation initiation factor (eIF2α), which then leads to the re-programming of the cell’s gene transcription and translation profile, and ultimately allowing cells to cope with and overcome starvation. For sensing starvation, Gcn2 must directly bind to its effector protein Gcn1. This interaction is mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Overexpression of the RWDBD alone impairs Gcn2 activation through disrupting endogenous Gcn1-Gcn2 interaction, hampering eIF2α phosphorylation, and consequently cells cannot overcome starvation and fail to grow.  This dominant negative phenotype is dependent on Arg-2259 in RWDBD.  Taking advantage of this phenotype in yeast, we here found that the dominant negative phenotype was reverted by each of four amino acid substitutions, K2270A, R2289A, R2297A, and K2301A.  This correlated with increased eIF2α phosphorylation levels, suggesting their relevance for Gcn2 activation.  All but Lys-2270 are fully surface exposed, suggesting that these amino acids may directly contact Gcn2.  We also found amino acid substitutions that enhanced the dominant negative phenotype of the overexpressed RWDBD, and correlated with further reduction in eIF2α-P levels.  Our findings suggest that two helices in Gcn1 constitute at least one Gcn2 contact point

A Rapid Extraction Method for mammalian cell cultures, suitable for quantitative immunoblotting analysis of proteins, including phosphorylated GCN2 and eIF2α

Many studies require the detection and relative quantitation of proteins from cell culture samples using immunoblotting. Limiting factors are the cost of protease inhibitors, the time required to break cells and generate samples, as well as the high risk of protein loss during cell breakage procedures. In addition, a common problem is the viscosity of lysed samples due to the released genomic DNA. As a consequence, the DNA needs to be broken down prior to denaturing polyacrylamide protein gel electrophoresis (SDS-PAGE), e.g. by passing the sample through a syringe gauge needle, sonication, or DNase treatment. In a quest to find a more cost-effective, fast, and yet robust procedure, we found that cell lysis, protein denaturation, and DNA fragmentation can be done in only two steps: harvesting followed by a simple non-laborious 2nd step. Similarly to many pre-existing cell breakage procedures, in our Rapid Protein Extraction (RPE) method, proteins liberated from cells are immediately exposed to a denaturing environment. However, advantages of our method are: • No breaking buffer is needed, instead proteins are liberated directly into the denaturing protein loading buffer used for SDS-PAGE. Consequently, our RPE method does not require any expensive inhibitors. • The RPE method does not involve post-lysis centrifugation steps; instead all cell material is dissolved during the 2nd step, the mixing-heat-treatment step which is new to this method. This prevents potential protein loss that may occur during centrifugation. In addition, this 2nd step simultaneously shears the genomic DNA, making an additional step for DNA fragmentation unnecessary. • The generated samples are suitable for high-quality quantitative immunoblotting. With our RPE method we successfully quantified the phosphorylated forms of protein kinase GCN2 and its substrate eIF2α. In fact, the western signals were stronger and with less background, as compared to samples generated with a pre-existing method. Method name: Rapid Protein Extraction Method (RPE method), Keywords: Mammalian cell culture, cell lysis, SDS-PAGE, quantitation, western blot, GCN2, eIF2 alph

Entwicklung eines Verfahrens zur routinemaessigen Aufnahme von Scherwellen

SIGLEDEGerman

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

Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics