Yeast p20 is a small, acidic protein that binds eIF4E, the cap-binding protein. It has been proposed to affect mRNA translation and degradation, however p20′s function as an eIF4E-binding protein (4E-BP) and its physiological significance has not been clearly established. In this paper we present data demonstrating that p20 is capable of binding directly to mRNA due to electrostatic interaction of a stretch of arginine and histidine residues in the protein with negatively charged phosphates in the mRNA backbone. This interaction contributes to formation of a ternary eIF4E/p20/capped mRNA complex that is more stable than complexes composed of capped mRNA bound to eIF4E in the absence of p20. eIF4E/p20 complex was found to have a more pronounced stimulatory effect on capped mRNA translation than purified eIF4E alone. Addition of peptides containing the eIF4E-binding domains present in p20 (motif YTIDELF), in eIF4G (motif YGPTFLL) or Eap1 (motif YSMNELY) completely inhibited eIF4E-dependent capped mRNA translation (in vitro), but had a greatly reduced inhibitory effect when eIF4E/p20 complex was present. We propose that the eIF4E/p20/mRNA complex serves as a stable depository of mRNAs existing in a dynamic equilibrium with other complexes such as eIF4E/eIF4G (required for translation) and eIF4E/Eap1 (required for mRNA degradation).

EIF4E, the mRNA cap-binding protein, is well known as a general initiation factor allowing for mRNA-ribosome interaction and cap-dependent translation in eukaryotic cells. In this review we focus on eIF4E and its interactors in unicellular organisms such as yeasts and protozoan eukaryotes. In a first part, we describe eIF4Es from yeast species such as Saccharomyces cerevisiae, Candida albicans, and Schizosaccharomyces pombe. In the second part, we will address eIF4E and interactors from parasite unicellular species—trypanosomatids and marine microorganisms—dinoflagellates. We propose that different strategies have evolved during evolution to accommodate cap-dependent translation to differing requirements. These evolutive “adjustments” involve various forms of eIF4E that are not encountered in all microorganismic species. In yeasts, eIF4E interactors, particularly p20 and Eap1 are found exclusively in Saccharomycotina species such as S. cerevisiae and C. albicans. For protozoan parasites of the Trypanosomatidae family beside a unique cap4-structure located at the 5′UTR of all mRNAs, different eIF4Es and eIF4Gs are active depending on the life cycle stage of the parasite. Additionally, an eIF4E-interacting protein has been identified in Leishmania major which is important for switching from promastigote to amastigote stages. For dinoflagellates, little is known about the structure and function of the multiple and diverse eIF4Es that have been identified thanks to widespread sequencing in recent years.

EIF4E, the cytoplasmatic cap-binding protein, is required for efficient cap-dependent translation. We have studied the influence of mutations that alter the activity and/or expression level of eIF4E on haploid and diploid cells in the yeast S. cerevisiae. Temperature-sensitive eIF4E mutants with reduced levels of expression and reduced cap-binding affinity clearly show a loss in haploid adhesion and diploid pseudohyphenation upon starvation for nitrogen. Some of these mutations affect the interaction of the cap-structure of mRNAs with the cap-binding groove of eIF4E. The observed reduction in adhesive and pseudohyphenating properties is less evident for an eIF4E mutant that shows reduced interaction with p20 (an eIF4E-binding protein) or for a p20-knockout mutant. Loss of adhesive and pseudohyphenating properties was not only observed for eIF4E mutants but also for knockout mutants of components of eIF4F such as eIF4B and eIF4G1. We conclude from these experiments that mutations that affect components of the eIF4F-complex loose properties such as adhesion and pseudohyphal differentiation, most likely due to less effective translation of required mRNAs for such processes.

A strain, which does not carry any auxotrophic markers, was transformed with knockout constructs for the genes HIS3 and ADE2 using the dominant antibiotic marker genes encoding for kanamycin/G418- and nourseothricin/NAT resistance. Thereby, homozygous derivatives that were histidine or adenine deficient were obtained. Histidine prototrophy was easily reconstituted by transforming his-defective diploid derivatives with yeast plasmids carrying the HIS3 gene. Despite different attempts, for example, by creating a rme1::KANX rme1::NATR double-deleted yeast strain (RME1 encodes for Regulator of Meiosis), no visible sporulation to obtain haploid derivatives could be obtained. Besides, no filamentation properties of were observed. As previously mentioned, this yeast strain was confirmed to thrive at 37°C, a temperature disliked by some but not all strains used in the laboratory. is a diploid derivative of that does not sporulates and survives at temperatures as those found in the human gut. It can be easily manipulated by using conventional yeast methods to introduce auxotrophic markers and obtain heterozygous diploid knockout derivatives that can be transformed with yeast plasmids following conventional yeast protocols, thereby it could be even suited for biochemical and genetic research purposes.

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae.