A Structural Domain Mediates Attachment of Ethanolamine Phosphoglycerol to Eukaryotic Elongation Factor 1A in Trypanosoma brucei

Ethanolamine phosphoglycerol (EPG) represents a protein modification that so far has only been found in eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is covalently attached to two conserved glutamate residues located in domains II and III of eEF1A. In contrast, Trypanosoma brucei eEF1A contains a single EPG attached to Glu362 in domain III. The sequence and/or structural requirements for covalent linkage of EPG to eEF1A have not been determined for any organism. Using a combination of biosynthetic labelling of parasites with tritiated ethanolamine and mass spectrometry analyses, we demonstrate that replacement of Glu362 in T. brucei eEF1A by site-directed mutagenesis prevents EPG attachment, whereas single or multiple amino acid substitutions around the attachment site are not critical. In addition, by expressing a series of eEF1A deletion mutants in T. brucei procyclic forms, we demonstrate that a peptide consisting of 80 amino acids of domain III of eEF1A is sufficient for EPG attachment to occur. Furthermore, EPG addition also occurs if domain III of eEF1A is fused to a soluble reporter protein. To our knowledge, this is the first report addressing amino acid sequence, or structure, requirements for EPG modification of eEF1A in any organism. Using T. brucei as a model organism, we show that amino acid substitutions around the modification site are not critical for EPG attachment and that a truncated version of domain III of eEF1A is sufficient to mediate EPG addition.     

Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae.

The functional domain structure of human DNA topoisomerase IIalpha and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase IIalpha and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.     

Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, I69M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, I69M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and I69M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to I69) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.     

Crystal structure of the his-tagged saccharopine reductase from Saccharomyces cerevisiae at 1.7-Å resolution

The three-dimensional structure of the saccharopine reductase enzyme from the budding yeast Saccharomyces cerevisiae was determined to 1.7-A resolution in the apo form by using molecular replacement. The enzyme monomer consists of three domains: domain I is a variant of the Rossmann fold, domain II folds into a alpha/beta structure containing a mixed seven-stranded beta-sheet as the central core, and domain III has an all-helical fold. Comparative fold alignment with the enzyme from Magnaporthe grisea suggests that domain I binds to NADPH, and domain II binds to saccharopine and is involved in dimer formation. Domain III is involved in closing the active site of the enzyme once substrates are bound. Structural comparison of the saccharopine reductase enzymes from S. cerevisiae and M. grisea indicates that domain II has the highest number of conserved residues, suggesting that it plays an important role in substrate binding and in spatially orienting domains I and III.     

Translation elongation factor-1A1 (eEF1A1) localizes to the spine by domain III

In vertebrates, there are two variants of eukaryotic peptide elongation factor 1A (eEF1A; formerly eEF-1α), eEF1A1 and eEF1A2, which have three well-conserved domains (D(I), D(II), and D(III)). In neurons, eEF1A1 is the embryonic type, which is expressed during embryonic development as well as the first two postnatal weeks. In the present study, EGFP-tagged eEF1A1 truncates were expressed in cortical neurons isolated from rat embryo (E18-19). Live cell images of transfected neurons showed that D(III)-containing EGFP-fusion proteins (EGFP-D(III), -D(II)-III, -D(I)-III) formed clusters that were confined within somatodendritic domains, while D(III)-missing ones (EGFP-D(I), -D(II), -D(I)-II) and control EGFP were homogeneously D(I)spersed throughout the neuron incluD(I)ng axons. In dendrites, EGFP-D(III) was targeted to the heads of spine- and filopoD(I)a-like protrusions, where it was colocalized with SynGAPα, a postsynaptic marker. Our data inD(I)cate that D(III) of eEF1A1 meD(I)ates formation of clusters and localization to spines.     

Fission yeast and a plant have functional homologues of the Sar1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast.

Sec12p and Sar1p are required for the formation of transport vesicles generated from the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. Sec12p is an ER type II membrane protein that mediates the membrane attachment of the GTP-binding Sar1 protein. The SAR1 gene is a multi-copy suppressor of a thermosensitive sec12 mutation. In an attempt to identify functional homologues of Sec12p and Sar1p from other eukaryotic organisms, we screened cDNA expression libraries derived from the fission yeast Schizosaccharomyces pombe and from the plant Arabidopsis thaliana for complementation of the sec12ts mutation. Four individual cDNAs were isolated, two of which encode the S. pombe and A. thaliana homologues of Sar1p. The three Sar1 proteins are 67% identical on average. The two other cDNAs encode type II membrane proteins which were designated Stl1p for the S. pombe protein and Stl2p for the A. thaliana protein (Stl stands for Sec12p-like). Both proteins have NH2-terminal cytoplasmic domains which resemble that of Sec12p: they are similar in size and present a significant degree of amino acid identity with the cytoplasmic domain of Sec12p. In contrast, the lumenal domains of Sec12p, Stl1p and Stl2p are very different in size and do not show any appreciable homology. That Stl1p and Stl2p are functional homologues of Sec12p was confirmed by showing that expression of either cloned gene complements a sec12 null mutation. Our results indicate that some of the mechanisms regulating vesicle formation at the ER are conserved not only in yeasts, but also in plants.     

Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein

Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation.     

Conservation of the prion properties of Ure2p through evolution

The yeast inheritable [URE3] element corresponds to a prion form of the nitrogen catabolism regulator Ure2p. We have isolated several orthologous URE2 genes in different yeast species: Saccharomyces paradoxus, S. uvarum, Kluyveromyces lactis, Candida albicans, and Schizosaccharomyces pombe. We show here by in silico analysis that the GST-like functional domain and the prion domain of the Ure2 proteins have diverged separately, the functional domain being more conserved through the evolution. The more extreme situation is found in the two S. pombe genes, in which the prion domain is absent. The functional analysis demonstrates that all the homologous genes except for the two S. pombe genes are able to complement the URE2 gene deletion in a S. cerevisiae strain. We show that in the two most closely related yeast species to S. cerevisiae, i.e., S. paradoxus and S. uvarum, the prion domains of the proteins have retained the capability to induce [URE3] in a S. cerevisiae strain. However, only the S. uvarum full-length Ure2p is able to behave as a prion. We also show that the prion inactivation mechanisms can be cross-transmitted between the S. cerevisiae and S. uvarum prions.     

The crystal structure of the glutathione S-transferase-like domain of elongation factor 1Bgamma from Saccharomyces cerevisiae

The crystal structure of the N-terminal 219 residues (domain 1) of the conserved eukaryotic translation elongation factor 1Bgamma (eEF1Bgamma), encoded by the TEF3 gene in Saccharomyces cerevisiae, has been determined at 3.0 A resolution by the single wavelength anomalous dispersion technique. The structure is overall very similar to the glutathione S-transferase proteins and contains a pocket with architecture highly homologous to what is observed in glutathione S-transferase enzymes. The TEF3-encoded form of eEF1Bgamma has no obvious catalytic residue. However, the second form of eEF1Bgamma encoded by the TEF4 gene contains serine 11, which may act catalytically. Based on the x-ray structure and gel filtration studies, we suggest that the yeast eEF1 complex is organized as an [eEF1A.eEF1Balpha.eEF1Bgamma]2 complex. A 23-residue sequence in the middle of eEF1Bgamma is essential for the stable dimerization of eEF1Bgamma and the quaternary structure of the eEF1 complex.     

Molecular docking uncovers TSPY binds more efficiently with eEF1A2 compared to eEF1A1

Testis-specific protein, Y-encoded (TSPY) binds to eukaryotic translation elongation factor 1 alpha (eEF1A) at its SET/NAP domain that is essential for the elongation during protein synthesis implicated with normal spermatogenesis. The eEF1A exists in two forms, eEF1A1 (alpha 1) and eEF1A2 (alpha 2), encoded by separate loci. Despite critical interplay of the TSPY and eEF1A proteins, literature remained silent on the residues playing significant roles during such interactions. We deduced 3D structures of TSPY and eEF1A variants by comparative modeling (Modeller 9.13) and assessed protein–protein interactions employing HADDOCK docking. Pairwise alignment using EMBOSS Needle for eEF1A1 and eEF1A2 proteins revealed high degree (~92%) of homology. Efficient binding of TSPY with eEF1A2 as compared to eEF1A1 was observed, in spite of the occurrence of significant structural similarities between the two variants. We also detected strong interactions of domain III followed by domains II and I of both eEF1A variants with TSPY. In the process, seven interacting residues of TSPY’s NAP domain namely, Asp 175, Glu 176, Asp 179, Tyr 183, Asp 240, Glu 244, and Tyr 246 common to both eEF1A variants were detected. Additionally, six lysine residues observed in eEF1A2 suggest their possible role in TSPY–eEF1A2 complex formation essential for germ cell development and spermatogenesis. Thus, more efficient binding of TSPY with eEF1A2 as compared to that of eEF1A1 established autonomous functioning of these two variants. Studies on mutated protein following similar approach would uncover the causative obstruction, between the interacting partners leading to deeper understanding on the structure–function relationship.     

真核翻译延伸因子1A蛋白家族功能位点的进化踪迹分析

真核翻译延伸因子1A（eEF1A）是真核生物蛋白质翻译过程中能将氨酰tRNA运送到核糖体A位点参与多肽延伸反应的多功能蛋白质. 本文主要利用多种生物信息学分析工具进行地中海涡虫翻译延伸因子1A（SmEF1A）蛋白序列的查找与eEF1A直系同源蛋白的搜索, 并基于90条直系同源蛋白进行eEF1A蛋白家族的进化踪迹分析和SmEF1A蛋白功能位点的比较研究. 结果表明,在eEF1A蛋白家族中共识别到338个踪迹残基位点和20个踪迹残基富集区域,SmEF1A蛋白的功能位点与踪迹残基位点密切相关,与GTP/Mg2+结合相关的S21、T72、D91、G94等重要位点均为全家族保守的踪迹残基,N 糖基化、磷酸化等蛋白修饰位点中踪迹残基位点往往是被修饰的部位或修饰功能发挥的关键辅助位点,而位于分子表面的配基结合口袋则与20个踪迹残基富集区域在分子表面形成的踪迹残基簇关系密切. eEF1A蛋白家族的进化踪迹分析为eEF1A蛋白重要功能区域关键残基的确定和未知功能位点的预测提供了重要信息.     

Elongation factor 1A is the target of growth inhibition in yeast caused by Legionella pneumophila glucosyltransferase Lgt1

Legionella is a pathogenic Gram-negative bacterium that can multiply inside of eukaryotic cells. It translocates numerous bacterial effector proteins into target cells to transform host phagocytes into a niche for replication. One effector of Legionella pneumophila is the glucosyltransferase Lgt1, which modifies serine 53 in mammalian elongation factor 1A (eEF1A), resulting in inhibition of protein synthesis and cell death. Here, we demonstrate that similar to mammalian cells, Lgt1 was severely toxic when produced in yeast and effectively inhibited in vitro protein synthesis. Saccharomyces cerevisiae strains, which were deleted of endogenous eEF1A but harbored a mutant eEF1A not glucosylated by Lgt1, were resistant toward the bacterial effector. In contrast, deletion of Hbs1, which is also an in vitro substrate of the glucosyltransferase, did not influence the toxic effects of Lgt1. Serial mutagenesis in yeast showed that Phe(54), Tyr(56) and Trp(58), located immediately downstream of serine 53 of eEF1A, are essential for the function of the elongation factor. Replacement of serine 53 by glutamic acid, mimicking phosphorylation, produced a non-functional eEF1A, which failed to support growth of S. cerevisiae. Our data indicate that Lgt1-induced lethal effect in yeast depends solely on eEF1A. The region of eEF1A encompassing serine 53 plays a critical role in functioning of the elongation factor.     

Either of the major H2A genes but not an evolutionarily conserved H2A.F/Z variant of Tetrahymena thermophila can function as the sole H2A gene in the yeast Saccharomyces cerevisiae.

H2A.F/Z histones are conserved variants that diverged from major H2A proteins early in evolution, suggesting they perform an important function distinct from major H2A proteins. Antisera specific for hv1, the H2A.F/Z variant of the ciliated protozoan Tetrahymena thermophila, cross-react with proteins from Saccharomyces cerevisiae. However, no H2A.F/Z variant has been reported in this budding yeast species. We sought to distinguish among three explanations for these observations: (i) that S. cerevisiae has an undiscovered H2A.F/Z variant, (ii) that the major S. cerevisiae H2A proteins are functionally equivalent to H2A.F/Z variants, or (iii) that the conserved epitope is found on a non-H2A molecule. Repeated attempts to clone an S. cerevisiae hv1 homolog only resulted in the cloning of the known H2A genes yHTA1 and yHTA2. To test for functional relatedness, we attempted to rescue strains lacking the yeast H2A genes with either the Tetrahymena major H2A genes (tHTA1 or tHTA2) or the gene (tHTA3) encoding hv1. Although they differ considerably in sequence from the yeast H2A genes, the major Tetrahymena H2A genes can provide the essential functions of H2A in yeast cells, the first such case of trans-species complementation of histone function. The Tetrahymena H2A genes confer a cold-sensitive phenotype. Although expressed at high levels and transported to the nucleus, hv1 cannot replace yeast H2A proteins. Proteins from S. cerevisiae strains lacking yeast H2A genes fail to cross-react with anti-hv1 antibodies. These studies make it likely that S. cerevisiae differs from most other eukaryotes in that it does not have an H2A.F/Z homolog. A hypothesis is presented relating the absence of H2A.F/Z in S. cerevisiae to its function in other organisms.     

Unique classes of mutations in the Saccharomyces cerevisiae G-protein translation elongation factor 1A suppress the requirement for guanine nucleotide exchange

G-proteins play critical roles in many cellular processes and are regulated by accessory proteins that modulate the nucleotide-bound state. Such proteins, including eukaryotic translation elongation factor 1A (eEF1A), are frequently reactivated by guanine nucleotide exchange factors (GEFs). In the yeast Saccharomyces cerevisiae, only the catalytic subunit of the GEF complex, eEF1Balpha, is essential for viability. The requirement for the TEF5 gene encoding eEF1Balpha can be suppressed by the presence of excess substrate, eEF1A. These cells, however, have defects in growth and translation. Two independent unbiased screens performed to dissect the cause of these phenotypes yielded dominant suppressors that bypass the requirement for extra eEF1A. Surprisingly, all mutations are in the G-protein eEF1A and cluster in its GTP-binding domain. Five mutants were used to construct novel strains expressing only the eEF1A mutant at normal levels. These strains show no growth defects and little to no decreases in total translation, which raises questions as to the evolutionary expression of GEF complexity and other potential functions of this complex. The location of the mutations on the eEF1A-eEF1Balpha structure suggests that their mechanism of suppression may depend on effects on the conserved G-protein elements: the P-loop and NKXD nucleotide-binding element.     

Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA.

Structural features of Internal Transcribed Spacer 1 (ITS1) that direct its removal from Saccharomyces cerevisiae pre-rRNA during processing were identified by an initial phylogenetic approach followed by in vivo mutational analysis of specific structural elements. We found that S. cerevisiae ITS1 can functionally be replaced by the corresponding regions from the yeasts Torulaspora delbrueckii, Kluyveromyces lactis and Hansenula wingei, indicating that structural elements required in cis for processing are evolutionarily conserved. Despite large differences in size, all ITS1 regions conform to the secondary structure proposed by Yeh et al. [Biochemistry 29 (1990) 5911-5918], showing five domains (I-V; 5'-->3') of which three harbour an evolutionarily highly conserved element. Removal of most of domain II, including its highly conserved element, did not affect processing. In contrast, highly conserved nucleotides directly downstream of processing site A2 in domain III play a major role in production of 17S, but not 26S rRNA. Domain IV and V are dispensable for 17S rRNA formation although an alternative, albeit inefficient, processing route to mature 17S rRNA may be mediated by a conserved region in domain IV. Each of these two domains is individually sufficient for efficient production of 26S rRNA, suggesting two independent processing pathways. We conclude that ITS1 is organized into two functionally and structurally distinct halves.     

A phylogenetically based secondary structure for the yeast telomerase RNA

BACKGROUND: Telomerase is a ribonucleoprotein complex whose RNA moiety dictates the addition of specific simple sequences onto chromosomes ends. While relevant for certain human genetic diseases, the contribution of the essential telomerase RNA to RNP assembly still remains unclear. Phylogenetic analyses of vertebrate and ciliate telomerase RNAs revealed conserved elements that potentially organize protein subunits for RNP function. In contrast, the yeast telomerase RNA could not be fitted to any known structural model, and the limited number of known sequences from Saccharomyces species did not permit the prediction of a yeast specific conserved structure. RESULTS: We cloned and analyzed the complete telomerase RNA loci (TLC1) from all known Saccharomyces species belonging to the "sensu stricto" group. Complementation analyses in S. cerevisiae and end mappings of mature RNAs ensured the relevance of the cloned sequences. By using phylogenetic comparative analysis coupled with in vitro enzymatic probing, we derived a secondary structure prediction of the Saccharomyces cerevisiae TLC1 RNA. This conserved secondary structure prediction includes a central domain that is likely to orchestrate DNA synthesis and at least two accessory domains important for RNA stability and telomerase recruitment. The structure also reveals a potential tertiary interaction between two loops in the central core. CONCLUSIONS: The predicted secondary structure of the TLC1 RNA of S. cerevisiae reveals a distinct folding pattern featuring well-separated but conserved functional elements. The predicted structure now allows for a detailed and rationally designed study to the structure-function relationships within the telomerase RNP-complex in a genetically tractable system.