An Extracellular/Membrane-Bound S100P Pool Regulates Motility and Invasion of Human Extravillous Trophoblast Lines and Primary Cells.

Whilst S100P has been shown to be a marker for carcinogenesis, we have shown, in non-physio-pathological states, that its expression promotes trophoblast motility and invasion but the mechanisms explaining these cellular processes are unknown. Here we identify the presence of S100P in the plasma membrane/cell surface of all trophoblast cells tested, whether lines, primary extravillous (EVT) cells, or section tissue samples using either biochemical purification of plasma membrane material, cell surface protein isolation through biotinylation, or microscopy analysis. Using extracellular loss of function studies, through addition of a specific S100P antibody, our work shows that inhibiting the cell surface/membrane-bound or extracellular S100P pools significantly reduces, but importantly only in part, both cell motility and cellular invasion in different trophoblastic cell lines, as well as primary EVTs. Interestingly, this loss in cellular motility/invasion did not result in changes to the overall actin organisation and focal adhesion complexes. These findings shed new light on at least two newly characterized pathways by which S100P promotes trophoblast cellular motility and invasion. One where cellular S100P levels involve the remodelling of focal adhesions whilst another, an extracellular pathway, appears to be focal adhesion independent. Both pathways could lead to the identification of novel targets that may explain why significant numbers of confirmed human pregnancies suffer complications through poor placental implantation.

Ezrin and Its Phosphorylated Thr567 Form Are Key Regulators of Human Extravillous Trophoblast Motility and Invasion.

The protein ezrin has been shown to enhance cancer cell motility and invasion leading to malignant behaviours in solid tumours, but a similar regulatory function in the early physiological reproduction state is, however, much less clear. We speculated that ezrin may play a key role in promoting first-trimester extravillous trophoblast (EVT) migration/invasion. Ezrin, as well as its Thr567 phosphorylation, were found in all trophoblasts studied, whether primary cells or lines. Interestingly, the proteins were seen in a distinct cellular localisation in long, extended protrusions in specific regions of cells. Loss-of-function experiments were carried out in EVT HTR8/SVneo and Swan71, as well as primary cells, using either ezrin siRNAs or the phosphorylation Thr567 inhibitor NSC668394, resulting in significant reductions in both cell motility and cellular invasion, albeit with differences between the cells used. Our analysis further demonstrated that an increase in focal adhesion was, in part, able to explain some of the molecular mechanisms involved. Data collected using human placental sections and protein lysates further showed that ezrin expression was significantly higher during the early stage of placentation and, importantly, clearly seen in the EVT anchoring columns, further supporting the potential role of ezrin in regulating migration and invasion in vivo.

The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis.

S100P protein is a potent inducer of metastasis in a model system, and its presence in cancer cells of patients is strongly associated with their reduced survival times. A well-established Furth Wistar rat metastasis model system, methods for measuring cell migration, and specific inhibitors were used to study pathways of motility-driven metastasis. Cells expressing C-terminal mutant S100P proteins display markedly-reduced S100P-driven metastasis in vivo and cell migration in vitro. These cells fail to display the low focal adhesion numbers observed in cells expressing wild-type S100P, and the mutant S100P proteins exhibit reduced biochemical interaction with non-muscle myosin heavy chain isoform IIA in vitro. Extracellular inhibitors of the S100P-dependent plasminogen activation pathway reduce, but only in part, wild-type S100P-dependent cell migration; they are without effect on S100P-negative cells or cells expressing C-terminal mutant S100P proteins and have no effect on the numbers of focal adhesions. Recombinant wild-type S100P protein, added extracellularly to S100P-negative cells, stimulates cell migration, which is abolished by these inhibitors. The results identify at least two S100P-dependent pathways of migration, one cell surface and the other intracellularly-linked, and identify its C-terminal lysine as a target for inhibiting multiple migration-promoting activities of S100P protein and S100P-driven metastasis.

Trophoblast uptake of DBP regulates intracellular actin and promotes matrix invasion.

Early pregnancy is characterised by elevated circulating levels of vitamin D binding protein (DBP). The impact of this on maternal and fetal health is unclear but DBP is present in the placenta, and DBP gene variants have been linked to malplacentation disorders such as preeclampsia. The functional role of DBP in the placenta was investigated using trophoblastic JEG3, BeWo and HTR8 cells. All three cell lines showed intracellular DBP with increased expression and nuclear localisation of DBP in cells treated with the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). When cultured in the serum of mice lacking DBP (DBP-/-), JEG3 cells showed no intracellular DBP indicating uptake of exogenous DBP. Inhibition of the membrane receptor for DBP, megalin, also suppressed intracellular DBP. Elimination of intracellular DBP with DBP-/- serum or megalin inhibitor suppressed matrix invasion by trophoblast cells and was associated with increased nuclear accumulation of G-actin. Conversely, treatment with 1,25D enhanced matrix invasion. This was independent of the nuclear vitamin D receptor but was associated with enhanced ERK phosphorylation, and inhibition of ERK kinase suppressed trophoblast matrix invasion. When cultured with serum from pregnant women, trophoblast matrix invasion correlated with DBP concentration, and DBP was lower in first-trimester serum from women who later developed preeclampsia. These data show that the trophoblast matrix invasion involves uptake of serum DBP and associated intracellular actin-binding and homeostasis. DBP is a potential marker of placentation disorders such as preeclampsia and may also provide a therapeutic option for improved placenta and pregnancy health.

Direct interaction of metastasis-inducing S100P protein with tubulin causes enhanced cell migration without changes in cell adhesion.

Overexpression of S100P promotes breast cancer metastasis in animals and elevated levels in primary breast cancers are associated with poor patient outcomes. S100P can differentially interact with nonmuscle myosin (NM) isoforms (IIA > IIC > IIB) leading to the redistribution of actomyosin filaments to enhance cell migration. Using COS-7 cells which do not naturally express NMIIA, S100P is now shown to interact directly with α,ß-tubulin in vitro and in vivo with an equilibrium Kd of 2-3 × 10-7 M. The overexpressed S100P is located mainly in nuclei and microtubule organising centres (MTOC) and it significantly reduces their number, slows down tubulin polymerisation and enhances cell migration in S100P-induced COS-7 or HeLa cells. It fails, however, to significantly reduce cell adhesion, in contrast with NMIIA-containing S100P-inducible HeLa cells. When taxol is used to stabilise MTs or colchicine to dissociate MTs, S100P's stimulation of migration is abolished. Affinity-chromatography of tryptic digests of α and ß-tubulin on S100P-bound beads identifies multiple S100P-binding sites consistent with S100P binding to all four half molecules in gel-overlay assays. When screened by NMR and ITC for interacting with S100P, four chemically synthesised peptides show interactions with low micromolar dissociation constants. The two highest affinity peptides significantly inhibit binding of S100P to α,ß-tubulin and, when tagged for cellular entry, also inhibit S100P-induced reduction in tubulin polymerisation and S100P-enhancement of COS-7 or HeLa cell migration. A third peptide incapable of interacting with S100P also fails in this respect. Thus S100P can interact directly with two different cytoskeletal filaments to independently enhance cell migration, the most important step in the metastatic cascade.

The anti-psychotic drug pimozide is a novel chemotherapeutic for breast cancer.

Pimozide, an antipsychotic drug of the diphenylbutylpiperidine class, has been shown to suppress cell growth of breast cancer cells in vitro. In this study we further explore the inhibitory effects of this molecule in cancer cells. We found that Pimozide inhibited cell proliferation in a dose- and time-dependent manner in MDA-MB-231 breast cancer cells and A549 lung cancer cells. Furthermore, we found that Pimozide also promoted apoptosis as demonstrated by cell cycle arrest and induction of double-strand DNA breaks but did not result in any effect in the non-transformed MCF10A breast cell line. In order to shed new lights into the molecular pathways affected by Pimozide, we show that Pimozide downregulated RAN GTPase and AKT at both protein and mRNA levels and inhibited the AKT signaling pathway in MDA-MB-231 breast cancer cells. Pimozide also inhibited the epithelial mesenchymal transition and cell migration and downregulated the expression of MMPs. Administration of Pimozide showed a potent in vivo antitumor activity in MDA-MB-231 xenograft animal model and reduced the number of lung metastases by blocking vascular endothelial growth factor receptor 2. Furthermore, Pimozide inhibited myofibroblast formation as evaluated by the reduction in α-smooth muscle actin containing cells. Thus, Pimozide might inhibit tumor development by suppressing angiogenesis and by paracrine stimulation provided by host reactive stromal cells. These results demonstrate a novel in vitro and in vivo antitumor activity of Pimozide against breast and lung cancer cells and provide the proof of concept for a putative Pimozide as a novel approach for cancer therapy.

S100P enhances the motility and invasion of human trophoblast cell lines.

S100P has been shown to be a marker for carcinogenesis where its expression in solid tumours correlates with metastasis and a poor patient prognosis. This protein's role in any physiological process is, however, unknown. Here we first show that S100P is expressed both in trophoblasts in vivo as well as in some corresponding cell lines in culture. We demonstrate that S100P is predominantly expressed during the early stage of placental formation with its highest expression levels occurring during the first trimester of gestation, particularly in the invading columns and anchoring villi. Using gain or loss of function studies through overexpression or knockdown of S100P expression respectively, our work shows that S100P stimulates both cell motility and cellular invasion in different trophoblastic and first trimester EVT cell lines. Interestingly, cell invasion was seen to be more dramatically affected than cell migration. Our results suggest that S100P may be acting as an important regulator of trophoblast invasion during placentation. This finding sheds new light on a hitherto uncharacterized molecular mechanism which may, in turn, lead to the identification of novel targets that may explain why significant numbers of confirmed human pregnancies suffer complications through poor placental implantation.

Microbial expression systems for membrane proteins.

Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.

Assessing Estrogen-Induced Proliferative Response in an Endometrial Cancer Cell Line Using a Universally Applicable Methodological Guide.

OBJECTIVE: Translational endometrial cancer (EC) research benefits from an in vitro experimental approach using EC cell lines. We demonstrated the steps that are required to examine estrogen-induced proliferative response, a simple yet important research question pertinent to EC, and devised a pragmatic methodological workflow for using EC cell lines in experimental models. METHODS: Comprehensive review of all commercially available EC cell lines was carried out, and Ishikawa cell line was selected to study the estrogen responsiveness with HEC1A, RL95-2, and MFE280 cell lines as comparators where appropriate, examining relevant differential molecular (steroid receptors) and functional (phenotype, anchorage-independent growth, hormone responsiveness, migration, invasion, and chemosensitivity) characteristics in 2-dimensional and 3-dimensional cultures in vitro using immunocytochemistry, immunofluorescence, quantitative polymerase chain reaction, and Western blotting. In vivo tumor, formation, and chemosensitivity were also assessed in a chick chorioallantoic membrane model. RESULTS: Short tandem repeat analysis authenticated the purchased cell lines, whereas gifted cells deviated significantly from the published profile. We demonstrate the importance of prior assessment of the suitability of each cell line for the chosen in vitro experimental technique. Prior establishment of baseline, nonenriched conditions was required to induce a proliferative response to estrogen. The chorioallantoic membrane model was a suitable in vivo multicellular animal model for EC for producing rapid and reproducible data. CONCLUSIONS: We have developed a methodological guide for EC researchers when using endometrial cell lines to answer important translational research questions (exemplified by estrogen-responsive cell proliferation) to facilitate robust data, while saving time and resources.

Vitamin D, the placenta and early pregnancy: effects on trophoblast function.

Pregnancy is associated with significant changes in vitamin D metabolism, notably increased maternal serum levels of active vitamin D, 1,25-dihydroxyvitamin (1,25(OH)2D). This appears to be due primarily to increased renal activity of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) that catalyzes synthesis of 1,25(OH)2D, but CYP27B1 expression is also prominent in both the maternal decidua and fetal trophoblast components of the placenta. The precise function of placental synthesis of 1,25(OH)2D remains unclear, but is likely to involve localized tissue-specific responses with both decidua and trophoblast also expressing the vitamin D receptor (VDR) for 1,25(OH)2D. We have previously described immunomodulatory responses to 1,25(OH)2D by diverse populations of VDR-expressing cells within the decidua. The aim of the current review is to detail the role of vitamin D in pregnancy from a trophoblast perspective, with particular emphasis on the potential role of 1,25(OH)2D as a regulator of trophoblast invasion in early pregnancy. Vitamin D deficiency is common in pregnant women, and a wide range of studies have linked low vitamin D status to adverse events in pregnancy. To date, most of these studies have focused on adverse events later in pregnancy, but the current review will explore the potential impact of vitamin D on early pregnancy, and how this may influence implantation and miscarriage.

Activation of tissue plasminogen activator by metastasis-inducing S100P protein.

S100P protein in human breast cancer cells is associated with reduced patient survival and, in a model system of metastasis, it confers a metastatic phenotype upon benign mammary tumour cells. S100P protein possesses a C-terminal lysine residue. Using a multiwell in vitro assay, S100P is now shown for the first time to exhibit a strong, C-terminal lysine-dependent activation of tissue plasminogen activator (tPA), but not of urokinase-catalysed plasminogen activation. The presence of 10 µM calcium ions stimulates tPA activation of plasminogen 2-fold in an S100P-dependent manner. S100P physically interacts with both plasminogen and tPA in vitro, but not with urokinase. Cells constitutively expressing S100P exhibit detectable S100P protein on the cell surface, and S100P-containing cells show enhanced activation of plasminogen compared with S100P-negative control cells. S100P shows C-terminal lysine-dependent enhancement of cell invasion. An S100P antibody, when added to the culture medium, reduced the rate of invasion of wild-type S100P-expressing cells, but not of cells expressing mutant S100P proteins lacking the C-terminal lysine, suggesting that S100P functions outside the cell. The protease inhibitors, aprotinin or α-2-antiplasmin, reduced the invasion of S100P-expressing cells, but not of S100P-negative control cells, nor cells expressing S100P protein lacking the C-terminal lysine. It is proposed that activation of tPA via the C-terminal lysine of S100P contributes to the enhancement of cell invasion by S100P and thus potentially to its metastasis-promoting activity.

Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin.

BACKGROUND: We previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 µg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5' untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10-80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A2aR) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described. RESULTS: Polysome profiling experiments were used to analyze the translational state of spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 µg/mL doxycycline); all but gcn5Δ were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31aΔ, rpl22aΔ, ssf1Δ and nop1Δ) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 µg/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2α. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5'UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3Δ or wild-type cells, suggesting that high-yielding strains are able to bypass 5'uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories. CONCLUSIONS: Regulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production.

Joining S100 proteins and migration: for better or for worse, in sickness and in health.

The vast diversity of S100 proteins has demonstrated a multitude of biological correlations with cell growth, cell differentiation and cell survival in numerous physiological and pathological conditions in all cells of the body. This review summarises some of the reported regulatory functions of S100 proteins (namely S100A1, S100A2, S100A4, S100A6, S100A7, S100A8/S100A9, S100A10, S100A11, S100A12, S100B and S100P) on cellular migration and invasion, established in both culture and animal model systems and the possible mechanisms that have been proposed to be responsible. These mechanisms involve intracellular events and components of the cytoskeletal organisation (actin/myosin filaments, intermediate filaments and microtubules) as well as extracellular signalling at different cell surface receptors (RAGE and integrins). Finally, we shall attempt to demonstrate how aberrant expression of the S100 proteins may lead to pathological events and human disorders and furthermore provide a rationale to possibly explain why the expression of some of the S100 proteins (mainly S100A4 and S100P) has led to conflicting results on motility, depending on the cells used.

Carnosine: can understanding its actions on energy metabolism and protein homeostasis inform its therapeutic potential?

The dipeptide carnosine (ß-alanyl-L-histidine) has contrasting but beneficial effects on cellular activity. It delays cellular senescence and rejuvenates cultured senescent mammalian cells. However, it also inhibits the growth of cultured tumour cells. Based on studies in several organisms, we speculate that carnosine exerts these apparently opposing actions by affecting energy metabolism and/or protein homeostasis (proteostasis). Specific effects on energy metabolism include the dipeptide's influence on cellular ATP concentrations. Carnosine's ability to reduce the formation of altered proteins (typically adducts of methylglyoxal) and enhance proteolysis of aberrant polypeptides is indicative of its influence on proteostasis. Furthermore these dual actions might provide a rationale for the use of carnosine in the treatment or prevention of diverse age-related conditions where energy metabolism or proteostasis are compromised. These include cancer, Alzheimer's disease, Parkinson's disease and the complications of type-2 diabetes (nephropathy, cataracts, stroke and pain), which might all benefit from knowledge of carnosine's mode of action on human cells.

Actin binding proteins: their ups and downs in metastatic life.

In order to metastasize away from the primary tumor site and migrate into adjacent tissues, cancer cells will stimulate cellular motility through the regulation of their cytoskeletal structures. Through the coordinated polymerization of actin filaments, these cells will control the geometry of distinct structures, namely lamella, lamellipodia and filopodia, as well as the more recently characterized invadopodia. Because actin binding proteins play fundamental functions in regulating the dynamics of actin polymerization, they have been at the forefront of cancer research. This review focuses on a subset of actin binding proteins involved in the regulation of these cellular structures and protrusions, and presents some general principles summarizing how these proteins may remodel the structure of actin. The main body of this review aims to provide new insights into how the expression of these actin binding proteins is regulated during carcinogenesis and highlights new mechanisms that may be initiated by the metastatic cells to induce aberrant expression of such proteins.

S100A4 downregulates filopodia formation through increased dynamic instability.

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin IIA, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration.

Understanding the yeast host cell response to recombinant membrane protein production.

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 α5ß1 integrin co-signaling.

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and ß1 integrin co-signaling pathway. By using α5 null cells, ß1 integrin functional blocking antibody, and a α5ß1 integrin targeting peptide A5-1, we demonstrate that the α5 and ß1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCα is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.

Improper organization of the actin cytoskeleton affects protein synthesis at initiation.

Although the actin cytoskeleton and the translation machinery are considered to be separate cellular complexes, growing evidence supports overlapping regulation of the two systems. Because of its interaction with actin, the eukaryotic translation elongation factor 1A (eEF1A) is proposed to be a regulator or link between these processes. Using a genetic approach with the yeast Saccharomyces cerevisiae, specific regions of eEF1A responsible for actin interactions and bundling were identified. Five new mutations were identified along one face of eEF1A. Dramatic changes in cell growth, cell morphology, and actin cable and patch formation as well as a unique effect on total translation in strains expressing the F308L or S405P eEF1A mutant form were observed. The translation effects do not correlate with reduced translation elongation but instead include an initiation defect. Biochemical analysis of the eEF1A mutant forms demonstrated reduced actin-bundling activity in vitro. Reduced total translation and/or the accumulation of 80S ribosomes in strains with either a mutation or a null allele of genes encoding actin itself or actin-regulating proteins Tpm1p, Mdm20p, and Bnirp/Bni1p was observed. Our data demonstrate that eEF1A, other actin binding proteins, and actin mutants affect translation initiation through the actin cytoskeleton.

Domain and nucleotide dependence of the interaction between Saccharomyces cerevisiae translation elongation factors 3 and 1A.

Eukaryotic translation elongation factor 3 (eEF3) is a fungal-specific ATPase proposed to catalyze the release of deacylated-tRNA from the ribosomal E-site. In addition, it has been shown to interact with the aminoacyl-tRNA binding GTPase elongation factor 1A (eEF1A), perhaps linking the E and A sites. Domain mapping demonstrates that amino acids 775-980 contain the eEF1A binding sites. Domain III of eEF1A, which is also involved in actin-related functions, is the site of eEF3 binding. The binding of eEF3 to eEF1A is enhanced by ADP, indicating the interaction is favored post-ATP hydrolysis but is not dependent on the eEF1A-bound nucleotide. A temperature-sensitive P915L mutant in the eEF1A binding site of eEF3 has reduced ATPase activity and affinity for eEF1A. These results support the model that upon ATP hydrolysis, eEF3 interacts with eEF1A to help catalyze the delivery of aminoacyl-tRNA at the A-site of the ribosome. The dynamics of when eEF3 interacts with eEF1A may be part of the signal for transition of the post to pre-translocational ribosomal state in yeast.