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.

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

The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction.

Asp56 in actin is critical for the full activity of the amino acid starvation-responsive kinase Gcn2.

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1-9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1-9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation-inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin-Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2.