Ribosomal Stalk Protein Research Articles

RNAi-Mediated Knockdown of Acidic Ribosomal Stalk Protein P1 Arrests Egg Development in Adult Female Yellow Fever Mosquitoes, Aedes aegypti.

After taking a blood meal, the fat body of the adult female yellow fever mosquito, Aedes aegypti, switches from a previtellogenic state of arrest to an active state of synthesizing large quantities of yolk protein precursors (YPPs) that are crucial for egg development. The synthesis of YPPs is regulated at both the transcriptional and translational levels. Previously, we identified the cytoplasmic protein general control nonderepressible 1 (GCN1) as a part of the translational regulatory pathway for YPP synthesis. In the current study, we used the C-terminal end of GCN1 to screen for protein-protein interactions and identified 60S acidic ribosomal protein P1 (P1). An expression analysis and RNAi-mediated knockdown of P1 was performed to further investigate the role of P1 in mosquito reproduction. We showed that in unfed (absence of a blood meal) adult A. aegypti mosquitoes, P1 was expressed ubiquitously in the mosquito organs and tissues tested. We also showed that the RNAi-mediated knockdown of P1 in unfed adult female mosquitoes resulted in a strong, transient knockdown with observable phenotypic changes in ovary length and egg deposition. Our results suggest that 60S acidic ribosomal protein P1 is necessary for mosquito reproduction and is a promising target for mosquito population control.

Mutational Analyses of the Cysteine-Rich Domain of Yvh1, a Protein Required for Translational Competency in Yeast.

Simple SummaryRibosome maturation in eukaryotes is a highly energy-consuming, evolutionarily conserved process, directed by a large number of auxiliary proteins (>200) called trans-acting factors (TAFs). TAFs direct the ordered and directional assembly of immature pre-40S and pre-60S complexes in multiple cellular compartments, but they are not themselves components of translationally competent ribosomes. Although the enzymatic properties of many TAFs are understood, others assist ribosome maturation without predicted enzymatic activity. An important late-acting 60S TAF is Mrt4, which initially binds to pre-60S complexes within the nucleus that must be released from pre-60S complexes following translocation into in the cytoplasm prior to the addition of a complex of acidic phosphoproteins denoting the ribosomal stalk, an essential component of the peptidyl transferase center (PTC) of ribosomes. Prior genetic studies implicated the dual-specificity phosphatase Yvh1 as the protein responsible for cytoplasmic Mrt4 removal, but how Yvh1 facilitates this is unknown. yvh1Δ strains are viable but grow slowly due to translational defects associated with improper ribosome assembly. Yvh1-dependent Mrt4 removal is independent of YVH1’s protein phosphatase domain but instead maps to an evolutionarily conserved cysteine-rich domain (CRD) of unknown function. In order to better understand how YVH1’s CRD facilitates Mrt4 removal and thus 60S maturation, we identified loss-of-function (LOF) variants of Yvh1 incapable of displacing Mrt4 precluding the addition of the ribosomal stalk. This approach additionally identified a variant of Yvh1 (Yvh1F283L) that functions as an expression-dependent, dominant-negative variant capable of perturbing ribosome assembly in cells containing wild-type YVH1. These findings are consistent with, and expand on, recent structural models for Yvh1-dependent Mrt4 removal from pre-60S complexes and generate novel first-generation probes that can be used to better understand eukaryotic ribosomal maturation.Ribosome assembly is a complex biological process facilitated by >200 trans-acting factors (TAFs) that function as scaffolds, place-holders or complex remodelers to promote efficient and directional ribosomal subunit assembly but are not themselves part of functional ribosomes. One such yeast TAF is encoded by Mrt4 which assembles onto pre-60S complexes in the nuclear compartment and remains bound to pre-60S complexes as they are exported into the cytoplasm. There, Mrt4 is displaced from pre-60S complexes facilitating the subsequent addition of the ribosomal stalk complex (P0/P1/P2). Ribosomal stalk proteins interact with translational GTPases (trGTPase) which facilitate and control protein synthesis on the ribosome. The rRNA-binding domain of Mrt4 is structurally similar to P0, with both proteins binding to the same interface of pre-60S subunits in a mutually exclusive manner; the addition of the ribosomal stalk therefore requires the displacement of Mrt4 from pre-60S subunits. Mrt4 removal requires the C-terminal cysteine-rich domain (CRD) of the dual-specificity phosphatase Yvh1. Unlike many other TAFs, yeast lacking Yvh1 are viable but retain Mrt4 on cytoplasmic pre-60S complexes precluding ribosomal stalk addition. Although Yvh1’s role in Mrt4 removal is well established, how Yvh1 accomplishes this is largely unknown. Here, we report an unbiased genetic screen to isolate Yvh1 variants that fail to displace Mrt4 from pre-60S ribosomes. Bioorthogonal non-canonical amino acid tagging (BONCAT) approaches demonstrate that these YVH1 loss-of-function variants also display defects in nascent protein production. The further characterization of one LOF variant, Yvh1F283L, establishes it as an expression-dependent, dominant-negative variant capable of interfering with endogenous Yvh1 function, and we describe how this Yvh1 variant can be used as a novel probe to better understand ribosome maturation and potentially ribosome heterogeneity in eukaryotes.

A Sixty-Year Research and Development of Trichosanthin, a Ribosome-Inactivating Protein.

Tian Hua Fen, a herbal powder extract that contains trichosanthin (TCS), was used as an abortifacient in traditional Chinese medicine. In 1972, TCS was purified to alleviate the side effects. Because of its clinical applications, TCS became one of the most active research areas in the 1960s to the 1980s in China. These include obtaining the sequence information in the 1980s and the crystal structure in 1995. The replication block of TCS on human immunodeficiency virus in lymphocytes and macrophages was found in 1989 and started a new chapter of its development. Clinical studies were subsequently conducted. TCS was also found to have the potential for gastric and colorectal cancer treatment. Studies on its mechanism showed TCS acts as an rRNA N-glycosylase (EC 3.2.2.22) by hydrolyzing and depurinating A-4324 in α-sarcin/ricin loop on 28S rRNA of rat ribosome. Its interaction with acidic ribosomal stalk proteins was revealed in 2007, and its trafficking in mammalian cells was elucidated in the 2000s. The adverse drug reactions, such as inducing immune responses, short plasma half-life, and non-specificity, somehow became the obstacles to its usage. Immunotoxins, sequence modification, or coupling with polyethylene glycerol and dextran were developed to improve the pharmacological properties. TCS has nicely shown the scientific basis of traditional Chinese medicine and how its research and development have expanded the knowledge and applications of ribosome-inactivating proteins.

Structural Distinctive 26SK, a Ribosome-Inactivating Protein from Jatropha curcas and Its Biological Activities.

Ribosome-inactivating proteins (RIPs) are a group of proteins exhibiting N-glycosidase activity leading to an inactivation of protein synthesis. Thirteen predicted Jatropha curcas RIP sequences could be grouped into RIP types 1 or 2. The expression of the RIP genes was detected in seed kernels, seed coats, and leaves. The full-length cDNA of two RIP genes (26SK and 34.7(A)SK) were cloned and studied. The 34.7(A)SK protein was successfully expressed in the host cells while it was difficult to produce even only a small amount of the 26SK protein. Therefore, the crude proteins were used from E. coli expressing 26SK and 34.7(A)SK constructs and they showed RIP activity. Only the cell lysate from 26SK could inhibit the growth of E. coli. In addition, the crude protein extracted from 26SK expressing cells displayed the effect on the growth of MDA-MB-231, a human breast cancer cell line. Based on in silico analysis, all 13 J. curcas RIPs contained RNA and ribosomal P2 stalk protein binding sites; however, the C-terminal region of the P2 stalk binding site was lacking in the 26SK structure. In addition, an amphipathic distribution between positive and negative potential was observed only in the 26SK protein, similar to that found in the anti-microbial peptide. These findings suggested that this 26SK protein structure might have contributed to its toxicity, suggesting potential uses against pathogenic bacteria in the future.

Structural insights into the Switching Off of the Interaction between the Archaeal Ribosomal Stalk and aEF1A by Nucleotide Exchange Factor aEF1B

The ribosomal stalk protein plays a crucial role in functional interactions with translational GTPase factors. It has been shown that the archaeal stalk aP1 binds to both GDP- and GTP-bound conformations of aEF1A through its C-terminal region in two different modes. To obtain an insight into how the aP1•aEF1A binding mode changes during the process of nucleotide exchange from GDP to GTP on aEF1A, we have analyzed structural changes in aEF1A upon binding of the nucleotide exchange factor aEF1B. The isolated archaeal aEF1B has nucleotide exchange ability in the presence of aa-tRNA but not deacylated tRNA, and increases activity of polyphenylalanine synthesis 4-fold. The aEF1B mutation, R90A, results in loss of its original nucleotide exchange activity but retains a remarkable ability to enhance polyphenylalanine synthesis. These results suggest an additional functional role for aEF1B other than in nucleotide exchange. The crystal structure of the aEF1A•aEF1B complex, resolved at 2.0 Å resolution, shows marked rotational movement of domain 1 of aEF1A compared to the structure of aEF1A•GDP•aP1, and this conformational change results in disruption of the original aP1 binding site between domains 1 and 3 of aEF1A. The loss of aP1 binding to the aEF1A•aEF1B complex was confirmed by native gel analysis. The results suggest that aEF1B plays a role in switching off the interaction between aP1 and aEF1A•GDP, as well as in nucleotide exchange, and promote translation elongation.

Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins.

The ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), which form the ancient ribosomal stalk, were discovered decades ago but their functions remain mysterious. We had previously shown that RPLP1/2 are exquisitely required for replication of dengue virus (DENV) and other mosquito-borne flaviviruses. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. We evaluated viral and cellular translation in RPLP1/2-depleted cells using ribosome profiling and found that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). We also find that RPLP1/2 depletion impacts a ribosome density for a small subset of cellular mRNAs. Importantly, the polarity of ribosomes on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, implying a role for RPLP1/2 in multi-pass transmembrane protein biogenesis. These analyses of viral and host RNAs converge to implicate RPLP1/2 as functionally important for ribosomes to elongate through ORFs encoding multiple TMDs. We suggest that the effect of RPLP1/2 at TMD associated pauses is mediated by improving the efficiency of co-translational folding and subsequent protein stability.

Structural and Functional Investigation and Pharmacological Mechanism of Trichosanthin, a Type 1 Ribosome-Inactivating Protein.

Trichosanthin (TCS) is an RNA N-glycosidase that depurinates adenine-4324 in the conserved α-sarcin/ricin loop (α-SRL) of rat 28 S ribosomal RNA (rRNA). TCS has only one chain, and is classified as type 1 ribosome-inactivating protein (RIP). Our structural studies revealed that TCS consists of two domains, with five conserved catalytic residues Tyr70, Tyr111, Glu160, Arg163 and Phe192 at the active cleft formed between them. We also found that the structural requirements of TCS to interact with the ribosomal stalk protein P2 C-terminal tail. The structural analyses suggest TCS attacks ribosomes by first binding to the C-terminal domain of ribosomal P protein. TCS exhibits a broad spectrum of biological and pharmacological activities including anti-tumor, anti-virus, and immune regulatory activities. This review summarizes an updated knowledge in the structural and functional studies and the mechanism of its multiple pharmacological effects.

The Interaction between the Ribosomal Stalk Proteins and Translation Initiation Factor 5B Promotes Translation Initiation.

Ribosomal stalk proteins recruit translation elongation GTPases to the factor-binding center of the ribosome. Initiation factor 5B (eIF5B in eukaryotes and aIF5B in archaea) is a universally conserved GTPase that promotes the joining of the large and small ribosomal subunits during translation initiation. Here we show that aIF5B binds to the C-terminal tail of the stalk protein. In the cocrystal structure, the interaction occurs between the hydrophobic amino acids of the stalk C-terminal tail and a small hydrophobic pocket on the surface of the GTP-binding domain (domain I) of aIF5B. A substitution mutation altering the hydrophobic pocket of yeast eIF5B resulted in a marked reduction in ribosome-dependent eIF5B GTPase activity in vitro In yeast cells, the eIF5B mutation affected growth and impaired GCN4 expression during amino acid starvation via a defect in start site selection for the first upstream open reading frame in GCN4 mRNA, as observed with the eIF5B deletion mutant. The deletion of two of the four stalk proteins diminished polyribosome levels (indicating defective translation initiation) and starvation-induced GCN4 expression, both of which were suppressible by eIF5B overexpression. Thus, the mutual interaction between a/eIF5B and the ribosomal stalk plays an important role in subunit joining during translation initiation in vivo.

The ribosomal stalk protein is crucial for the action of the conserved ATPase ABCE1.

The ATP-binding cassette (ABC) protein ABCE1 is an essential factor in ribosome recycling during translation. However, the detailed mechanochemistry of its recruitment to the ribosome, ATPase activation and subunit dissociation remain to be elucidated. Here, we show that the ribosomal stalk protein, which is known to participate in the actions of translational GTPase factors, plays an important role in these events. Biochemical and crystal structural data indicate that the conserved hydrophobic amino acid residues at the C-terminus of the archaeal stalk protein aP1 binds to the nucleotide-binding domain 1 (NBD1) of aABCE1, and that this binding is crucial for ATPase activation of aABCE1 on the ribosome. The functional role of the stalk•ABCE1 interaction in ATPase activation and the subunit dissociation is also investigated using mutagenesis in a yeast system. The data demonstrate that the ribosomal stalk protein likely participates in efficient actions of both archaeal and eukaryotic ABCE1 in ribosome recycling. The results also show that the stalk protein has a role in the function of ATPase as well as GTPase factors in translation.

Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association

The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12-IF2 interaction on Escherichia coli ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12-IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners-all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.

Rigid Rod Model for the Disordered Domains of Ribosomal Stalk Proteins P1P2

In eukaryotes, the ribosomal stalk proteins, which are responsible for the recruitment of transcription factors, consist of two P1P2 heterodimers attached onto one copy of P0. Recent NMR structure of full-length P1P2 reveals that their C-terminals are disordered in nature and can extend up to 125A away from the center of the heterodimer. Here, we report a rigid rod model aimed at sampling the structures of the disordered C-terminal tails of P1P2. With sequence-dependent angles and dihedrals, our model is constructed from all-atom (AA) molecular dynamics (MD) simulations of short P1P2 segments. Despite its apparent simplicity, the end-to-end distance and radius of gyration computed with our model are found to be in decent agreement with extensive AA simulations of the entire disordered C-terminals of P1P2. Using our model, we further investigate the experimentally observed correlation between the recruitment efficiency of the ribosome inactivating proteins and the length of P1P2 C-terminal tails. Overall, results of our calculation are in good agreement with experimental measurements based on truncation and insertion studies of P1P2, indicating that our rigid rod model offers an efficient and promising approach in structural and functional study of the disordered domains in the ribosome stalk proteins.

Transcriptomic profile of cystic fibrosis patients identifies type I interferon response and ribosomal stalk proteins as potential modifiers of disease severity.

Cystic Fibrosis (CF) is the most common monogenic disease among people of Western European descent and caused by mutations in the CFTR gene. However, the disease severity is immensely variable even among patients with similar CFTR mutations due to the possible effect of ‘modifier genes’. To identify genetic modifiers, we applied RNA-seq based transcriptomic analyses in CF patients with a mild and severe lung phenotype. Global gene expression and enrichment analyses revealed that genes of the type I interferon response and ribosomal stalk proteins are potential modifiers of CF related lung dysfunction. The results provide a new set of CF modifier genes with possible implications as new therapeutic targets for the treatment of CF.

Binding of translation elongation factors to individual copies of the archaeal ribosomal stalk protein aP1 assembled onto aP0

Ribosomes in all organisms contain oligomeric and flexible proteins called stalks, which are responsible for the recruitment of translational GTPase factors to the ribosome. Archaeal ribosomes have three stalk homodimers (aP1)2 that constitute a heptameric complex with the anchor protein aP0. We investigated the factor binding ability of aP1 proteins assembled onto aP0, by gel-retardation assays. The isolated aP0(aP1)2(aP1)2(aP1)2 complex, as well as the form bound to the Escherichia coli 50S core, as a hybrid 50S particle, interacted strongly with elongation factor aEF2, but weakly with aEF1A. These interactions were disrupted by a point mutation, F107S, at the C-terminus of aP1. To examine the ability of each copy of aP0-associated aP1 to bind to elongation factors, we constructed aP0·aP1 variant trimers, composed of an aP0 mutant and a single (aP1)2 dimer. Biochemical and quantitative analyses revealed that the resultant three trimers, aP0(aP1)2I, aP0(aP1)2II, and aP0(aP1)2III, individually bound two molecules of aEF2, suggesting that each copy of the aP1 C-terminal region in the aP0·aP1 trimers can bind tightly to aEF2. Interestingly, the unstable binding of aEF1A to each of the three aP0·aP1 trimers was remarkably stabilized in the presence of aEF2. The stability of the aEF1A binding to the stalk complex may be affected by the presence of aEF2 bound to the complex, by an unknown mechanism.

Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2.

Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.

Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

Structures of eukaryotic ribosomal stalk proteins and its complex with trichosanthin, and their implications in recruiting ribosome-inactivating proteins to the ribosomes.

Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA.

Molecular insights into the interaction of the ribosomal stalk protein with elongation factor 1α.

In all organisms, the large ribosomal subunit contains multiple copies of a flexible protein, the so-called ‘stalk’. The C-terminal domain (CTD) of the stalk interacts directly with the translational GTPase factors, and this interaction is required for factor-dependent activity on the ribosome. Here we have determined the structure of a complex of the CTD of the archaeal stalk protein aP1 and the GDP-bound archaeal elongation factor aEF1α at 2.3 Å resolution. The structure showed that the CTD of aP1 formed a long extended α-helix, which bound to a cleft between domains 1 and 3 of aEF1α, and bridged these domains. This binding between the CTD of aP1 and the aEF1α•GDP complex was formed mainly by hydrophobic interactions. The docking analysis showed that the CTD of aP1 can bind to aEF1α•GDP located on the ribosome. An additional biochemical assay demonstrated that the CTD of aP1 also bound to the aEF1α•GTP•aminoacyl-tRNA complex. These results suggest that the CTD of aP1 interacts with aEF1α at various stages in translation. Furthermore, phylogenetic perspectives and functional analyses suggested that the eukaryotic stalk protein also interacts directly with domains 1 and 3 of eEF1α, in a manner similar to the interaction of archaeal aP1 with aEF1α.

Ribosomal protein P2 localizes to the parasite zoite-surface and is a target for invasion inhibitory antibodies in Toxoplasma gondii and Plasmodium falciparum

In the malarial parasite Plasmodium falciparum, the conserved ribosomal stalk protein P2 (PfP2) exhibits extra-ribosomal stage-specific oligomerization and trafficking to the host red cell membrane. Antibodies directed against PfP2 arrested cell division. We sought to examine whether P2 from a closely related Apicomplexan parasite, Toxoplasma gondii, exhibits similar properties in terms of its oligomeric status as well as such unique host-cell localization. Circular dichroism spectroscopy of recombinant P2 from T. gondii (TgP2) showed a structure similar to that of PfP2, but unlike PfP2, which forms SDS- and DTT-resistant oligomers, TgP2 exhibited only a weak SDS-resistant dimerization. Also, unlike PfP2 localization to the infected erythrocyte surface, TgP2 did not localize to the host membrane in T. gondii infected human foreskin fibroblast cells. However, P2 protein was detected on the free tachyzoite surface, corroborated by localization of epitope-tagged P2 transfected in T. gondii. The presence of P2 on the surface of P. falciparum merozoites was also observed, and specific antibodies raised against the P2 protein blocked both T. gondii and P. falciparum zoite invasion of the host cells. Thus, although certain moonlighting functions of the acidic ribosomal protein P2 are different amongst P. falciparum and T. gondii, the P2 protein localizes to the surface of the invasive zoite form, and appears to constitute a potential target for host cell invasion inhibition in both the Apicomplexan infections.

Cytoskeletal proteins associate with components of the ribosomal maturation and translation apparatus in Xenopus stage I oocytes.

Actin-based cytoskeleton (CSK) and microtubules may bind to RNAs and related molecules implicated in translation. However, many questions remain to be answered regarding the role of cytoskeletal components in supporting the proteins involved in steps in the maturation and translation processes. Here, we performed co-immunoprecipitation and immunofluorescence to examine the association between spectrins, keratins and tubulin and proteins involved in 60S ribosomal maturation and translation in Xenopus stage I oocytes, including ribosomal rpl10, eukaryotic initiation factor 6 (Eif6), thesaurins A/B, homologs of the eEF1α elongation factor, and P0, the ribosomal stalk protein. We found that rpl10 and eif6 cross-reacted with the actin-based CSK and with tubulin. rpl10 co-localizes with spectrin, particularly in the perinuclear region. eif6 is similarly localized. Given that upon ribosomal maturation, the insertion of rpl10 into the 60S subunit occurs simultaneously with the release of eif6, one can hypothesise that actin-based CSK and microtubules provide the necessary scaffold for the insertion/release of these two molecules and, subsequently, for eif6 transport and binding to the mature 60S subunit. P0 and thesaurins cross-reacted with only spectrin and cytokeratins. Thesaurins aggregated at the oocyte periphery, rendering this a territory favourable site for protein synthesis; the CSK may support the interaction between thesaurins and sites of the translating ribosome. Moreover, given that the assembly of the ribosome stalk, where P0 is located, to the 60S subunit is essential for the release of eif6, it can be hypothesised that the CSK can facilitate the binding of the stalk to the 60S.

The Acidic Ribosomal Stalk Proteins Are Not Required for the Highly Specific Inactivation Exerted by α-Sarcin of the Eukaryotic Ribosome

The ribosomal sarcin/ricin loop (SRL) is the target of ribosome-inactivating proteins like the N-glycosidase ricin and the fungal ribotoxin α-sarcin. The eukaryotic ribosomal stalk directly interacts with several members of the N-glycosidase family, favoring their disruption of the SRL. Here we tested this hypothesis for the ribotoxin α-sarcin. Experiments with isolated ribosomes, cell-free translation systems, and viability assays with Saccharomyces cerevisiae strains defective in acidic stalk proteins showed that the inactivation exerted by α-sarcin is independent of the composition of the ribosomal stalk. Therefore, α-sarcin, with the same ribosomal target as ricin, seems to access the SRL by a different pathway.