Translational regulation by ABC systems.

Elongation factor 3 is a cytosolic protein required by the fungal ribosomes for in vitro protein synthesis and for in vivo growth. EF-3 stimulates binding of EF-1:GTP:aa-tRNA ternary complex to the ribosomal A site by facilitated release of the deacylated tRNA from the E site. The reaction requires ATP hydrolysis. EF-3 contains two ATP binding sequence (NBS) motifs. NBSI is sufficient for the intrinsic ATPase activity. NBSII is essential for the ribosome-stimulated functions.

Evolutionary divergence of an elongation factor 3 from Cryptococcus neoformans.

Elongation factor 3 (EF3) is considered a promising drug target for the control of fungal diseases because of its requirement for protein synthesis and survival of fungi and a lack of EF3 in the mammalian host. However, EF3 has been characterized only in ascomycete yeast. In order to understand the role of EF3 in a basidiomycete yeast, we cloned the gene encoding EF3 from Cryptococcus neoformans (CnEF3), an important fungal pathogen in immunocompromised patients, including those infected with human immunodeficiency virus. CnEF3 was found to encode a 1,055-amino-acid protein and has 44% identity with EF3 from Saccharomyces cerevisiae (YEF3). Expressed CnEF3 exhibited ATPase activity that was only modestly stimulated by ribosomes from S. cerevisiae. In contrast, CnEF3 showed tight binding to cryptococcal ribosomes, as shown by an inability to be removed under conditions which successfully remove Saccharomyces EF3 from ribosomes (0.5 M KCl or 2 M LiCl). CnEF3 also poorly complemented a YEF3 defect in a diploid null mutant and two temperature-sensitive mutants which have been shown previously to be complemented well by EF3 from other ascomycetes, such as Candida albicans. These data clearly identify the presence of a functioning EF3 in the basidiomycete yeast C. neoformans, which demonstrates an evolutionary divergence from EF3 of ascomycete yeast.

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 A resolution.

Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome small middle dotEF2 complex at 17.5 A resolution by three-dimensional (3D) cryo-electron microscopy. EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli. However, pronounced differences were found in two prominent regions. First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive. Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit. The shape and position of domain IV of EF2 suggest that it might interact directly with P-site-bound tRNA.

Limited proteolysis of yeast elongation factor 3. Sequence and location of the subdomains.

Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi. We used limited proteolysis to study the organization of the subdomains of EF-3. Trypsinolysis of EF-3 at 30 degrees C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the large fragments from further degradation. ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments. The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding. Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3. Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA. Neither tRNA nor rRNA altered the tryptic cleavage pattern. The proteolytic products were sequenced by mass spectrometric analysis. EF-3 is blocked NH(2)-terminally by an acetylated serine. The 90-, 70-, and 50-kDa fragments are also blocked NH(2)-terminally, confirming their origin. The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with no known function. The 70-kDa fragment (Ser(2)-Lys(668)) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain. The primary ribosome binding site is located near the loosely structured carboxyl-terminal end.

Functional interaction of yeast elongation factor 3 with yeast ribosomes.

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a monomeric protein with a molecular mass of 116,000. EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth. The protein stimulates the binding of EF-1 alpha :GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site. The reaction requires ATP hydrolysis. EF-3 contains two ATP-binding sequence motifs (NBS). NBSI is sufficient for the intrinsic ATPase function. NBSII is essential for ribosome-stimulated activity. By limited proteolysis, EF-3 was divided into two distinct functional domains. The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity. The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes. The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA. We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end. Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein.

Yeast elongation factor 3: structure and function.

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a single polypeptide protein with a molecular weight of 116,000 required by yeast ribosomes for in vitro translation and for in vivo growth. The YEF3 gene, located on chromosome xii, is essential for the survival of yeast. The deduced amino acid sequence of EF-3 has revealed the presence of duplicated ATP-binding cassettes similar to those present in the membrane associated transporters. The carboxy-terminus of EF-3 contains blocks of lysine boxes essential for its functional interaction with yeast ribosomes. EF-3 stimulates binding of aminoacyl-tRNA to the ribosomal A-site by facilitating release of deacylated tRNA from the exit site (E-site). Chasing experiments revealed that EF-3 enhances the rate of tRNA dissociation from the E-site by a factor of two without affecting the affinity of the site for tRNA. EF-3 function is dependent on ATP hydrolysis. The existence of functional homologs of EF-3 in higher eukaryotes is still an open question. Further investigations are needed to settle this issue.

Competition and cooperation amongst yeast elongation factors.

Elongation factor 3 (EF-3) is an essential requirement for translation in fungi. We previously reported activation of EF-3-ATPase by yeast ribosomes. EF-3 interacts with both ribosomal subunits and shows high affinity for 60S subparticles. Translational inhibitors alpha-sarcin, ricin and auto-immune antibodies to GTPase-activation center inhibit binding of EF-2 but not of EF-3 to yeast ribosomes. EF-2 competes with EF-3 for the ribosomal binding sites and inhibits EF-3-ATPase activity. Neomycin relieves the inhibitory effect of EF-2 on EF-3 function. The apparent competition between EF-2 and EF-3 may represent binding of these two proteins to specific conformational states of the ribosome. EF-3 stimulates ternary complex binding to yeast ribosomes. Neither the binding of EF-3 to ribosomes, nor the ribosome-dependent EF-3-ATPase activity are influenced by EF-1 alpha. Three lines of experimental evidence suggest a direct interaction between EF-1 alpha and EF-3. A polyclonal antibody to EF-3 immunoprecipitates EF-1 alpha along with EF-3. EF-1 alpha co-migrates with GST-EF-3 on glutathione-Sepharose columns. ELISA tests demonstrate an interference of EF-3/anti-EF-3 interaction by EF-1 alpha but not by EF-2. These results strongly suggest that the stimulatory effect of EF-3 on the ternary complex binding to yeast ribosomes involves a direct interaction between EF-1 alpha and EF-3.

Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2.

In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.

Involvement of a 50-kDa mRNP protein from Saccharomyces cerevisiae in mRNA binding to ribosomes.

A yeast 50-kDa mRNA-binding protein (50mRNP) is found selectively associated with the 48S and 80S initiation complexes. This protein is structurally related to the translational elongation factor EF-1alpha. The protein reacts with antibodies directed against EF-1alpha and, similarly, EF-1alpha recognizes antibodies against the 50mRNP protein. This is evidence that they share at least one epitope which allows a similar antigenic behavior. In addition, both proteins show similar cleavage patterns upon treatment with the endoproteinase Lys-C. A murine antibody raised against 50mRNP inhibits both 48S and 80S initiation complex formation. The inhibitory effect is relieved by preincubating anti-50mRNP with EF-1alpha. Antibody to EF-1alpha manifests a similar inhibitory pattern for the formation of 48S and 80S complexes. These data strongly suggest that 50mRNP is an EF-1alpha-like polypeptide essential for the formation of the above complexes.

Overexpression and purification of elongation factor 3 from Saccharomyces cerevisiae.

The translational elongation factor 3 (EF-3) from Saccharomyces cerevisiae was overexpressed and purified to near homogeneity from the post-ribosomal supernatant fraction (S-100). A detailed protocol for the isolation of overexpressed EF-3 is presented. The procedure involves ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose and affinity chromatography on ATP-agarose. A protein purity of > or = 96% was established by quantitating the silver-stained SDS/polyacrylamide gel. The present method facilitates isolation of EF-3 in large amounts in high yield.

Functional subdomains of yeast elongation factor 3. Localization of ribosome-binding domain.

Elongation factor 3 (EF-3) is an essential requirement of the fungi for translational elongation. EF-3 is an ATPase, and the hydrolytic activity is stimulated 2 orders of magnitude by yeast ribosomes. Limited trypsinolysis of EF-3 results in the cleavage of a single peptide bond between residues 774 (Arg) and 775 (Gln), generating polypeptides of approximate molecular mass 90 and 30 kDa. The 90-kDa fragment is relatively resistant to proteolysis and retains ribosome-independent ATPase activity. The 30-kDa fragment is further proteolyzed into smaller fragments and retains the specificity for binding to yeast ribosomes. Both the intact EF-3 and the 30-kDa fragment are protected from proteolysis by yeast ribosomes. EF-3 is NH2 terminally blocked, and so is the 90-kDa fragment. The COOH terminally derived 30-kDa fragment contains glutamine (residue 775) at the NH2-terminal end. A construct was designed representing the COOH-terminal domain of EF-3 (30-kDa fragment), subcloned, and expressed as a glutathione S-transferase fusion in yeast. The glutathione S-transferase-30-kDa peptide remains stringently associated with ribosomes. Isolated fusion peptide rebinds to yeast ribosomes with high affinity. Based on these results, we propose that at least one of the ribosome-binding sites of EF-3 resides at the COOH-terminal end of the protein.

Interaction of yeast elongation factor 3 with polynucleotides, ribosomal RNA and ribosomal subunits.

In addition to the two usual eukaryotic elongation factors (EF-1 alpha and EF-2) fungal ribosomes need a third protein, elongation factor 3, for translation. EF-3 is essential for in vivo and in vitro protein synthesis. Functionally, EF-3 stimulates EF-1 alpha dependent binding of aminoacyl-tRNA to the ribosomal A site when E site is occupied by deacylated tRNA. EF-3 has intrinsic ATPase activity which is regulated by the functional state of the ribosome. EF-3 ATPase is activated by both 40S and 60S ribosomal subunits. However intact 80S ribosomes are needed for efficient activation of EF-3 ATPase. EF-3 appears to be an RNA binding protein with high affinity for polynucleotides containing guanosine rich sequences. To determine whether guanosine rich sequence of ribosomal RNA is involved in EF-3 binding, an antisense oligonucleotide dC6 was used to block EF-3 interaction with the ribosome. The oligonucleotide suppresses activation of EF-3 ATPase by 40S ribosomal subunit and not by the 60S or the 80S particles. Poly(U)-directed polyphenylalanine synthesis by yeast ribosomes is inhibited by dC6. To define the binding site of the oligonucleotide and presumably of EF-3 on 18S ribosomal RNA, hydrolysis of rRNA by RNase H was followed in the presence of dC6. These experiments reveal an RNase H cleavage site at 1094GGGGGG1099 sequence of 18S ribosomal RNA. This guanosine rich sequence of rRNA is suggested to be involved in EF-3 binding to yeast ribosome. Data presented in this communication suggest that the activity of EF-3 involved a direct interaction with the guanosine rich sequence of rRNA.

The elongation factor 3 unique in higher fungi and essential for protein biosynthesis is an E site factor.

Two elongation factors drive the ribosomal elongation cycle; elongation factor 1 alpha (EF-1 alpha) mediates the binding of an aminoacyl-tRNA to the ribosomal A site, whereas elongation factor 2 (EF-2) catalyzes the translocation reaction. Ribosomes from yeast and other higher fungi require a third elongation factor (EF-3) which is essential for the elongation process, but the step affected by EF-3 has not yet been identified. Here we demonstrate that the first and the third tRNA binding site (A and E sites, respectively) of yeast ribosomes are reciprocally linked; if the A site is occupied the E site has lost its binding capability, and vice versa, if the E site is occupied the A site has a low affinity for tRNAs. EF-3 is essential for EF-1 alpha-dependent A site binding of amino-acyl-tRNA only when the E site is occupied with a deacylated tRNA. The ATP-dependent activity of EF-3 is required for the release of deacylated tRNA from the E site during A site occupation.

Comparative analysis of ATPase of yeast elongation factor 3 and ATPase associated with Tetrahymena ribosomes.

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. The biochemical function of EF-3 has recently been defined. The protein removes deacylated tRNA from the ribosomal exit site (E-site) thus facilitating occupation of the ribosomal A-site by aa-tRNA. A functional homolog of yeast EF-3 has not been identified in non-fungal species. Yeast EF-3 is a ribosome-dependent ATPase that can also accept GTP and ITP as substrates. The function of EF-3 in ribosomal reactions requires ATP hydrolysis. An ATPase activity associated with higher eukaryotic ribosomes has been claimed to be a direct functional homolog of yeast EF-3. Comparative analysis of biochemical, immunological and functional properties of ATPase activity associated with the ribosomes isolated from the ciliated protozoan Tetrahymena pyriformis with that of yeast EF-3 ATPase indicates that these two activities are significantly different. Results reported in this communication strongly suggest that the ribosome associated ATP hydrolytic activity of Tetrahymena pyriformis is not a functional homolog of yeast EF-3.

Comparative analysis of ribosome-associated adenosinetriphosphatase (ATPase) from pig liver and the ATPase of elongation factor 3 from Saccharomyces cerevisiae.

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational machinery. Non-fungal organisms do not have and do not require a soluble form of the third elongation factor for translation. To test whether non-fungal organisms have a direct analog of EF-3 incorporated in the structure of the ribosomes, a comparison of EF-3 adenosinetriphosphatase (ATPase) with ATPases associated with pig liver ribosomes was carried out. EF-3 function depends on ATP (GTP) hydrolysis. The hydrolytic activity of EF-3 is enhanced by two orders of magnitude by yeast ribosomes due to an increase in the turnover rate of EF-3. The nucleotide hydrolytic activity of EF-3 is significantly constrained by the binding of aminoacylated tRNA(Phe) to poly(U)-programmed ribosomes. The translational inhibitors--neomycin and alpha-sarcin suppress the ATPase activity of EF-3. These results reflect a direct correlation between EF-3 ATPase and the functional state of the ribosome. Four lines of evidence indicate that yeast EF-3 ATPase is functionally distinct from pig liver ribosome associated ATPases. The kinetic parameters of ATPases from these two sources are different. Poly(U) and tRNA have no effect on the ATPase activity associated with the pig liver ribosomes. The latter activity is unaffected by the translational inhibitors neomycin and alpha-sarcin. The translational activity of pig liver ribosomes is not influenced by ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate. In an in vitro system, one can demonstrate a small but consistent stimulatory effect of yeast EF-3 on polyphenylalanine synthesis by pig liver ribosomes only when EF-1 alpha is present at a limited concentration. The EF-3 effect disappears when EF-1 alpha is added in a stoichiometric amount to the pig liver ribosomes. This result is in contrast to the yeast system where the ribosomes are completely dependent on EF-3 at all concentrations of EF-1 alpha.

Protein synthesis elongation factor EF-1 alpha is essential for ubiquitin-dependent degradation of certain N alpha-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu.

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin pathway involves diverse recognition signals and distinct enzymatic factors. A few proteins are recognized via their N-terminal amino acid residue and conjugated by a ubiquitin-protein ligase that recognizes this residue. Most substrates, including the N alpha-acetylated proteins that constitute the vast majority of cellular proteins, are targeted by different signals and are recognized by yet unknown ligases. We have previously shown that degradation of N-terminally blocked proteins requires a specific factor, designated FH, and that the factor acts along with the 26S protease complex to degrade ubiquitin-conjugated proteins. Here, we demonstrate that FH is the protein synthesis elongation factor EF-1 alpha. (a) Partial sequence analysis reveals 100% identity to EF-1 alpha. (b) Like EF-1 alpha, FH binds to immobilized GTP (or GDP) and can be purified in one step using the corresponding nucleotide for elution. (c) Guanine nucleotides that bind to EF-1 alpha protect the ubiquitin system-related activity of FH from heat inactivation, and nucleotides that do not bind do not exert this effect. (d) EF-Tu, the homologous bacterial elongation factor, can substitute for FH/EF-1 alpha in the proteolytic system. This last finding is of particular interest since the ubiquitin system has not been identified in prokaryotes. The activities of both EF-1 alpha and EF-Tu are strongly and specifically inhibited by ubiquitin-aldehyde, a specific inhibitor of ubiquitin isopeptidases. It appears, therefore, that EF-1 alpha may be involved in releasing ubiquitin from multiubiquitin chains, thus rendering the conjugates susceptible to the action of the 26S protease complex.

Transfer RNA binding to 80S ribosomes from yeast: evidence for three sites.

The number of tRNA binding sites in 80S ribosomes from Saccharomyces cerevisiae was assessed by means of tRNA saturation and translocation experiments. In the absence of cognate mRNA yeast ribosomes could bind 0.6 [32P]tRNA(Phe) per 80S while poly(U) programmed ribosomes accepted up to 1.7 tRNA(Phe) molecules per 80S or 0.5 molecules of Ac[14C]Phe-tRNA(Phe) per 80S. Compared with the known features of E. coli ribosomes these binding values indicated both the presence of three tRNA binding sites and the validity of the exclusion principle for peptidyl-tRNA binding to yeast ribosomes. Upon EF-2 dependent translocation of a complex containing deacyl-tRNA in the P-site and AcPHe-tRNA in the A-site, the deacylated tRNA does not leave the ribosome quantitatively. This observation suggests the presence of an E site in 80S ribosomes which is functionally equivalent to the one previously characterized in prokaryotic systems.

Discrimination between initiation and elongation of protein biosynthesis in yeast: identity assured by a nucleotide modification in the initiator tRNA.

Cytoplasmic initiator tRNAs from plants and fungi possess an unique 2'-phosphoribosyl residue at position 64 of their sequence. In yeast tRNA(iMet), this modified nucleotide located in the T-stem of the tRNA is a 2'-1''-(beta-O-ribofuranosyl-5''-phosphoryl)-adenosine. The phosphoribosyl residue of this modified nucleoside was removed chemically by treatment involving periodate oxidation of tRNA(iMet) and regeneration of the 3'-terminal adenosine with ATP (CTP):tRNA nucleotidyl transferase. The role of phosphoribosylation at position 64 for interaction with elongation factor eEF-1 alpha and initiation factor 2 (eIF-2) was investigated in the homologous yeast system. Whereas the 5'-phosphoribosyl residue prevents the binding of Met-tRNA(iMet) to eEF-1 alpha, it does not influence the interaction with eIF-2. After removal of the ribosyl group, the demodified initiator tRNA showed binding to eEF-1 alpha, but no change was detected with respect to the interaction with the initiation factor eIF-2. This observation is interpreted to mean that a single modification of an eucaryotic initiator tRNA in yeast serves as a negative discriminant for eEF-1 alpha, thus preventing the initiator tRNA(iMet) from entering the elongation cycle of protein biosynthesis.

Characterization of yeast EF-1 alpha: non-conservation of post-translational modifications.

Elongation factor 1 alpha (EF-1 alpha) is an abundant cellular protein and its amino-acid sequence has been inferred from numerous organisms, including bacteria, archaebacteria, plants and animals. In large measure, it would appear that the overall structure has probably been maintained given the 33% identity and 56% similarity of Escherichia coli EF-Tu with human EF-1 alpha. Chemical sequencing of EF-Tu and EF-1 alpha has revealed that these proteins are post-translationally modified. In order to assess the possible function of these modifications, we have chemically sequenced the EF-1 alpha from the lower eukaryote Saccharomyces cerevisiae (yeast). To our surprise, the methylation pattern of yeast EF-1 alpha was quite different from either rabbit or brine shrimp EF-1 alpha with only the trimethyllysine at position 79 conserved although the yeast protein is 81% identical to rabbit EF-1 alpha. A dimethyllysine was observed at position 316 which corresponds to a trimethyllysine in brine shrimp and rabbit EF-1 alpha. The other positions in yeast EF-1 alpha which were methylated were unrelated to the other six possible positions for modification observed in brine shrimp or rabbit EF-1 alpha. In addition, the unique glyceryl-phosphorylethanolamine observed in mammalian EF-1 alpha and suspected in brine shrimp EF-1 alpha was not found in yeast EF-1 alpha.