Protein Translation Elongation Research Articles

Structural basis of bacterial protein translation elongation

Recently, better understanding of ribosome function has been achieved through structural determination. These results provided a wealth of information on how genetic codes are precisely translated into proteins that control life, as well as established the platform to put ribosome as one of the richest validated targets for antibacterial drug discovery. We discovered a new ribosome crystal form obtained from ribosome lacking subunit L9, which allows crystallization of the ribosome in the presence of elongation factors Tu and G (EF-Tu/EF-G), trapped by antibiotics kirromycin and fusidic acid, respectively. These structures for the first time at atomic level offer insight into the molecular mechanism of protein elongation, which lies at the heart of translation. In each elongation cycle, EF-G facilitates the movement of tRNA-mRNA by one codon, which is coupled to the ratchet-like rotation of the ribosome complex and is triggered by EF-G-mediated GTP hydrolysis. The structure of pre-translocational ribosome bound to EF-G trapped with a GTP analogue, sheds light on how the positioning of the catalytic residue His87 into the active site is coupled to the hydrophobic gate opening which involves the sarcin-ricin loop (SRL) and domain III of EF-G. These results provide the structural basis for the GTPase activation of EF-G. After translocation, a cognate deacylated tRNA can only move together with the codon into the ribosomal E site. We also determined the structure of a cognate tRNA bound to the ribosomal E site, representing an authentic ribosome elongation complex. Quality control in protein translation by bacterial toxin has been associated with ribosome stalling and rescue system that is critical for cell to adapt environmental stress. Finally, I will introduce our structural progress on bacterial toxin bound to ribosome towards understanding of a classical acid-base catalysis mechanism.

Identification of AKT kinases as unfavorable prognostic factors for hepatocellular carcinoma by a combination of expression profile, interaction network analysis and clinical validation

identification of key markers that differentiate occurrence and progression of hepatocellular carcinoma (HCC) is of great significance to develop novel prognostic factors and improve therapeutic strategies. The aim of this study was to screen novel markers for HCC by combining expression profile, interaction network analysis and clinical validation. HCC significant molecules which were differentially expressed in HCC tissues were obtained from five existing HCC related databases (OncoDB.HCC, HCC.net, dbHCCvar, EHCO and Liverome). The protein-protein interaction network of HCC significant proteins was constructed and 331 candidate HCC markers were identified by calculating four topological features of the network ('Degree', 'Betweenness', 'Closeness' and 'K-coreness'). According to the enrichment analysis on Gene ontology items and KEGG pathways, these candidate HCC markers were more frequently involved in cellular protein metabolic processes, translational elongation and intracellular signaling cascade, which are associated with cancer development and metastasis. Among 331 candidate HCC markers, the three AKT kinase family members (AKT1-AKT3) were selected for clinical validation by immunohistochemistry analysis using 130 HCC specimens and matched adjacent non-neoplastic liver tissues. Interestingly, the upregulation of AKT1, AKT2 and AKT3 proteins were all significantly associated with tumor aggressiveness and poor prognosis in patients with HCC. this study provided an integrated analysis by combining expression profile and interaction network analysis to identify a list of biologically significant HCC related markers and pathways. Further experimental validation also indicated that AKT1, AKT2 and AKT3 proteins may all be novel unfavorable prognostic factors for patients with HCC.

Quantitative proteomic analysis of sphere-forming stem-like oral cancer cells

IntroductionThe purpose of this study is to identify target proteins that may play important functional roles in oral cancer stem-like cells (CSCs) using mass spectrometry-based quantitative proteomics.MethodsSphere-formation assays were performed on highly invasive UM1 and lowly invasive UM2 oral cancer cell lines, which were derived from the same tongue squamous cell carcinoma, to enrich CSCs. Quantitative proteomic analysis of CSC-like and non-CSC UM1 cells was carried out using tandem mass tagging and two-dimensional liquid chromatography with Orbitrap mass spectrometry.ResultsCSC-like cancer cells were found to be present in the highly invasive UM1 cell line but absent in the lowly invasive UM2 cell line. Stem cell markers SOX2, OCT4, SOX9 and CD44 were up-regulated, whereas HIF-1 alpha and PGK-1 were down-regulated in CSC-like UM1 cells versus non-CSC UM1 cells. Quantitative proteomic analysis indicated that many proteins in cell cycle, metabolism, G protein signal transduction, translational elongation, development, and RNA splicing pathways were differentially expressed between the two cell phenotypes. Both CREB-1-binding protein (CBP) and phosphorylated CREB-1 were found to be significantly over-expressed in CSC-like UM1 cells.ConclusionsCSC-like cells can be enriched from the highly invasive UM1 oral cancer cell line but not from the lowly invasive UM2 oral cancer cell line. There are significant proteomic alterations between CSC-like and non-CSC UM1 cells. In particular, CBP and phosphorylated CREB-1 were significantly up-regulated in CSC-like UM1 cells versus non-CSC UM1 cells, suggesting that the CREB pathway is activated in the CSC-like cells.

A Pentapeptide Monocyte Locomotion Inhibitory Factor Protects Brain Ischemia Injury by Targeting the eEF1A1/Endothelial Nitric Oxide Synthase Pathway

Ischemic stroke is a major cause of death worldwide but lacks viable treatment or treatment targets. Monocyte locomotion inhibitory factor (MLIF) is a small heat-stable pentapeptide produced by Entamoeba histolytica in axenic culture, which is supposed to protect the brain from ischemic injury; the mechanism, however, remains unknown. In this study, we further investigated the mechanism underlying the protective role of MLIF in brain ischemia. A middle cerebral artery occlusion model in rats was used for detecting the effect of MLIF in the brain ischemia in vivo. To identify targets of MLIF in brain endothelial cells, we performed immunoprecipitation of biotin-conjugated MLIF and mass spectrometry. MLIF can protect the brain from ischemic injury in vivo, yielding decreased ischemic volume, prolonged survival, and improved neurological outcome. In vitro studies showed that MLIF displayed protective effects through inhibition of expression of pathological inflammatory adhesion molecules and enhancing endothelial nitric oxide synthase expression and nitric oxide release in the cerebrovascular endothelium. The target screening experiments demonstrated binding of MLIF to the ribosomal protein translation elongation factor eEF1A1. MLIF enhanced endothelial nitric oxide synthase expression through stabilization of endothelial nitric oxide synthase mRNA, and eEF1A1 was shown to be necessary for this enhanced expression. Knockdown of eEF1A1 or inhibition of endothelial nitric oxide synthase attenuated MLIF-mediated inhibition of adhesion molecule expression. In this study, we identified a new potential pharmacologically targetable mechanism underlying MLIF's protective effects in brain ischemia through the eEF1A1/endothelial nitric oxide synthase pathway.

Characterization of Two Malaria Parasite Organelle Translation Elongation Factor G Proteins: The Likely Targets of the Anti-Malarial Fusidic Acid

Malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. One such drug is fusidic acid, which inhibits the translation component elongation factor G. The response of P. falciparum to fusidic acid was characterised using extended SYBR-Green based drug trials. This revealed that fusidic acid kills in vitro cultured P. falciparum parasites by immediately blocking parasite development. Two bacterial-type protein translation elongation factor G genes are identified as likely targets of fusidic acid. Sequence analysis suggests that these proteins function in the mitochondria and apicoplast and both should be sensitive to fusidic acid. Microscopic examination of protein-reporter fusions confirm the prediction that one elongation factor G is a component of parasite mitochondria whereas the second is a component of the relict plastid or apicoplast. The presence of two putative targets for a single inhibitory compound emphasizes the potential of elongation factor G as a drug target in malaria.

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factorviamitogen-activated protein kinase

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)- p21Cip/WAF1 activation, and suppressed by the mitogenactivated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

Complexome of Escherichia coli cytosolic proteins under normal native conditions

The interactions between proteins are important for the majority of biological functions and the interacting proteins are usually assembled into a complex. Knowing a set of protein complexes of a cell (complexome) is, therefore, essential for a better understanding and global view of cell functions. To visualize and identify the protein complexome of E. coli K-12 under normal native conditions on a proteome-wide scale, we developed an integrated proteomic platform with the combination of 2-D native/SDS-PAGE-based proteomics with co-immunoprecipitation, far-Western blotting, His-tag affinity purification and functional analysis, and used it to investigate the E. coli cytosolic complexome. A total of 24 distinct heteromeric and 8 homomeric protein complexes were identified. These complexes mainly contributed to glycolysis/gluconeogenesis, bioinformation processing, and cellular processes. Of the 24 hetereomeric complexes, 16 were reported for the first time, and 2 known complexes contained novel components that have not been reported previously based on DIP database search. Among them, RpoC-RpsA-Tig-GroL was found to be involved in transcriptional and co-translational folding, and EF-G-TufA-Tsf-RpsA linked a protein synthesis site with protein translational elongation factors. This systematic proteome analysis provides new insights into E. coli molecular systems biology.

Protein Markers for Insulin-Producing Beta Cells with Higher Glucose Sensitivity

Background and MethodologyPancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins.Principal FindingsAll tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain.ConclusionsQuantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes.

Mechanical stretch activates signaling events for protein translation initiation and elongation in C2C12 myoblasts.

It has been proposed that mechanically induced tension is the critical factor in the induction of muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, the effect of mechanical stretch on intracellular signaling for protein translation initiation and elongation was studied in C2C12 myoblasts. Cells were grown on a silicone elastomer chamber and subjected to 30-min of 5 or 15% constant static or cyclic (60 cycles/min) uniaxial stretch. Western blot analyses revealed that p70 S6 kinase (p70S6K) and eukaryotic elongation factor 2 (eEF2), which are the markers for translation initiation and peptide chain elongation, respectively, were activated by both static and cyclic stretch. The magnitude of activation was greater in response to the 15% cyclic stretch. Cyclic stretch also increased the phosphorylation of MAP kinases (p38 MAPK, ERK1/2 and JNK). However, the pharmacological inhibition of MAP kinases did not block the stretch-induced activation of p70S6K and eEF2. An inhibitor of the mammalian target of rapamycin (mTOR) blocked the stretch-induced phosphorylation of p70S6K but did not affect the eEF2 activation. A broad-range tyrosine kinase inhibitor, genistein, blocked the stretch-induced activation of p70S6K and eEF2, whereas Src tyrosine kinase and Janus kinase (JAK) inhibitors did not. These results suggest that the stretch-induced activation of protein translation initiation and elongation in mouse myoblast cell lines is mediated by tyrosine kinase(s), except for Src kinase or JAK.

Mechanical stretch induces activation of eukaryotic elongation factor 2 in C2C12 myoblast

It has been proposed that mechanically‐induced tension was the critical event for initiating muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, effect of mechanical stretch on the protein translation initiation is studied in C2C12 myoblast. C2C12 cells were plated on fibronectin‐coated silicone elastomer chamber in DMEM containing 10% fetal bovine serum. Cells were grown in CO2 incubator and subjected to 30 min of cyclic 15% biaxial stretch at a frequency of 1Hz. 30 min of cyclic stretch increased the phosphorylation levels of p38 mitogen activated protein kinase (MAPK) and extracellular‐regulated kinase 1 and 2 (ERK1/2). Eukaryotic elongation factor 2 (eEF2), a marker for peptide chain elongation process, was also activated (dephosphorylated) by cyclic stretch. These results suggest that p38 MAPK and/or ERK1/2 pathway may be involved in stretch‐induced activation of protein translation elongation process. This study is supported by Grant‐in‐Aid for Scientific Research C (20500577 to N.N.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Proteomic Study for Responses to Cadmium Stress in Rice Seedlings

A proteomic approach including two-dimensional electrophoresis and mass spectrometric (MALDI-TOF MS) analyses was used to investigate the responses to cadmium (Cd) stress in seedlings of rice ( Oryza sativa L.) varieties Shanyou 63 and Aizaizhan. Cd stress significantly inhibited root and shoot growth, and affected the global proteome in rice roots and leaves, which induced or upregulated the expression of corresponding proteins in rice roots and leaves when rice seedlings were exposed to 0.1 or 1.0 mmol/L Cd. The Cd-induced proteins are involved in chelation and compartmentation of Cd, elimination of active oxygen free radicals, detoxification of toxic substances, degradation of denatured proteins or inactivated enzymes, regulation of physiologic metabolism and induction of pathogenesis-related proteins. Comparing the Cd-induced proteins between the two varieties, the β-glucosidase and pathogenesis-related protein family 10 proteins were more drastically induced by Cd stress in roots and leaves of Aizaizhan, and the UDP-glucose protein transglucosylase and translational elongation factor Tu were induced by 0.1 mmol/L Cd stress in roots of Shanyou 63. This may be one of the important mechanisms for higher tolerance to Cd stress in Shanyou 63 than in Aizaizhan.

Targeting eEF1A by aLegionella pneumophilaeffector leads to inhibition of protein synthesis and induction of host stress response

The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1Bgamma, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1Bgamma. We also show that infection by L. pneumophila leads to eEF1A-mediated activation of the heat shock regulatory protein HSF1 in a virulence-dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila-infected cells.

Binding of Elongation Factor eEF1A2 to Phosphatidylinositol 4-Kinase β Stimulates Lipid Kinase Activity and Phosphatidylinositol 4-Phosphate Generation

Eukaryotic protein translation elongation factor 1 alpha 2 (eEF1A2) is an oncogene that transforms mammalian cell lines and increases their tumorigenicity in nude mice. Increased expression of eEF1A2 occurs during the development of breast, ovarian, and lung cancer. Here, we report that eEF1A2 directly binds to and activates phosphatidylinositol 4-kinase III beta (PI4KIIIbeta), an enzyme that converts phosphatidylinositol to phosphatidylinositol 4-phosphate. Purified recombinant eEF1A2 increases PI4KIIIbeta lipid kinase activity in vitro, and expression of eEF1A2 in rat and human cells is sufficient to increase overall cellular phosphatidylinositol 4-kinase activity and intracellular phosphatidylinositol 4-phosphate abundance. siRNA-mediated reduction in eEF1A2 expression concomitantly reduces phosphatidylinositol 4-kinase activity. This identifies a physical and functional relationship between eEF1A2 and PI4KIIIbeta.

Structural basis of yeast aminoacyl-tRNA synthetase complex formation revealed by crystal structures of two binary sub-complexes

The yeast aminoacyl-tRNA synthetase (aaRS) complex is formed by the methionyl- and glutamyl-tRNA synthetases (MetRS and GluRS, respectively) and the tRNA aminoacylation cofactor Arc1p. It is considered an evolutionary intermediate between prokaryotic aaRS and the multi- aaRS complex found in higher eukaryotes. While a wealth of structural information is available on the enzymatic domains of single aaRS, insight into complex formation between eukaryotic aaRS and associated protein cofactors is missing. Here we report crystal structures of the binary complexes between the interacting domains of Arc1p and MetRS as well as those of Arc1p and GluRS at resolutions of 2.2 and 2.05 Å, respectively. The data provide a complete structural model for ternary complex formation between the interacting domains of MetRS, GluRS and Arc1p. The structures reveal that all three domains adopt a glutathione S-transferase (GST)-like fold and that simultaneous interaction of Arc1p with GluRS and MetRS is mediated by the use of a novel interface in addition to a classical GST dimerization interaction. The results demonstrate a novel role for this fold as a heteromerization domain specific to eukaryotic aaRS, associated proteins and protein translation elongation factors.

Recombination between elongation factor 1alpha genes from distantly related archaeal lineages.

Homologous recombination (HR) and lateral gene transfer are major processes in genome evolution. The combination of the two processes, HR between genes in different species, has been documented but is thought to be restricted to very similar sequences in relatively closely related organisms. Here we report two cases of interspecific HR in the gene encoding the core translational protein translation elongation factor 1alpha (EF-1alpha) between distantly related archaeal groups. Maximum-likelihood sliding window analyses indicate that a fragment of the EF-1alpha gene from the archaeal lineage represented by Methanopyrus kandleri was recombined into the orthologous gene in a common ancestor of the Thermococcales. A second recombination event appears to have occurred between the EF-1alpha gene of the genus Methanothermobacter and its ortholog in a common ancestor of the Methanosarcinales, a distantly related euryarchaeal lineage. These findings suggest that HR occurs across a much larger evolutionary distance than generally accepted and affects highly conserved essential "informational" genes. Although difficult to detect by standard whole-gene phylogenetic analyses, interspecific HR in highly conserved genes may occur at an appreciable frequency, potentially confounding deep phylogenetic inference and hypothesis testing.

Effect of acidic pH on expression of surface-associated proteins of Streptococcus oralis.

Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.

Analysis of Streptococcus mutans proteins modulated by culture under acidic conditions.

Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.

In situ immunocytochemical evidence that a homolog of protein translation elongation factor EF-1? is associated with microtubules in carrot cells

Evidence indicates that elongation factor-1α (EF-1α), a ubiquitous and abundant protein factor involved in the first step of peptide elongation, is also associated with the cytoskeleton in a variety of organisms. Although the effects of these associations on EF-1α's translational function have not been examined, the associations do appear to result in non-passive effects on the cytoskeleton. A carrot homolog of EF-1α, pp 50, has been reported to interact with microtubules in vitro, inducing the formation of microtubule bundles that can be dissociated by Ca2+/calmodulin. The characterization of anti-pp 50 antibodies is reported here. Immunocytochemistry, using anti-pp 50 and anti-tubulin antibodies, was used to investigate the co-localization of pp 50 and microtubules in situ. In carrot protoplasts fixed after detergent lysis, at least a fraction of pp 50 appears to be associated with microtubules. Treatment of such protoplasts with amiprophos-methyl (APM) reduced both the presence of microtubules and the co-localizing pp 50-associated fluorescence. In taxol-treated protoplasts, increases in both microtubules and the colocalizing pp 50-associated fluorescence were observed. When carrot protoplasts were fixed prior to detergent extraction, confocal laser scanning microscopy likewise revealed co-localization. Furthermore, what is likely to be a fluorescence resonance energy transfer (FRET) between fluorochromes associated with anti-pp 50 and anti-tubulin reporters was observed, indicating that some pp 50 is intimately associated with microtubules. The in situ cytoarchitectural evidence is consistent with a function previously proposed for pp 50 based on in vitro experiments — that pp 50 is a plant microtubuleassociated protein (MAP) whose function can be modulated by a Ca2+/calmodulin signal transduction mechanism in plant cells.

Multifunctional Proteins in Tetrahymena: 14-nm Filament Protein/Citrate Synthase and Translation Elongation Factor-1α

One gene encoding a protein has been shown to have two entirely different functions. Such a phenomenon, which has been called "gene sharing," was first known in crystallins. We found two multifunctional proteins in the ciliated protozoan Tetrahymena: 14-nm filament protein and protein translation elongation factor 1-alpha (EF-1 alpha). The 14-nm filament protein has dual functions as a citrate synthase in mitochondria and as a cytoskeletal protein in cytoplasm. In cytoplasm, the 14-nm filament protein was involved in oral morphogenesis and in pronuclear behavior during conjugation. The observation that Tetrahymena intramitochondrial filamentous inclusions contain the 14-nm filament protein and that the citrate synthase activity of the 14-nm filament protein is decreased by polymerization and increased by depolymerization, suggests a possible modulating mechanism of citrate synthase activity by monomer-polymer conversion in mitochondria in situ. The EF-1 alpha functions as an F-actin-bundling protein and a 14-nm filament-associated protein as well as an elongation factor in protein synthesis. The F-actin-bundling activity of EF-1 alpha was regulated by Ca2+ and calmodulin. Here we review the properties and functions of two multifunctional proteins in Tetrahymena.

Chlorophyll regulates accumulation of the plastid-encoded chlorophyll proteins P700 and D1 by increasing apoprotein stability.

Chlorophyll protein accumulation in barley (Hordeum vulgare L.) chloroplasts is controlled posttranscriptionally by light-induced formation of chlorophyll a. The abundance of translation initiation complexes associated with psbA, psaA, and rbcL mRNAs was measured using extension and inhibition analysis in plants grown in the dark for 4.5 d and then illuminated for up to 16 h. Light-induced accumulation of the chlorophyll proteins was not accompanied by changes in the abundance of translation initiation complexes, indicating that regulation of chlorophyll protein accumulation at this stage of development does not occur at the level of translation initiation. Translational runoff assays were performed in the presence of lincomycin, an inhibitor of translation initiation, to determine whether chlorophyll protein accumulation was regulated at the level of translation elongation. The extent of ribosome runoff of psaA and psbA mRNAs was similar in the presence or absence of chlorophyll, indicating that chlorophyll did not alter chlorophyll protein translation elongation. Polysome-associated D1 translation intermediates were radiolabeled in the presence or absence of chlorophyll, even though full-length D1 accumulated only in the presence of chlorophyll. Chlorophyll influenced the stability of D1 translation intermediates to a small extent and greatly increased D1 stability after release from ribosomes. Overall, these results demonstrate that light-induced chlorophyll biosynthesis triggers the accumulation of the chlorophyll proteins D1 and P700 in barley chloroplasts by enhancement of chlorophyll apoprotein stability.