Trigger factor depletion or overproduction causes defective cell division but does not block protein export.

Trigger factor is an abundant cytosolic protein of Escherichia coli which can stabilize proOmpA for in vitro translocation across inner membrane vesicles. The gene encoding E. coli trigger factor was isolated and sequenced, allowing construction of strains in which the expression of trigger factor is readily regulated. We found no defect in the in vivo rate of synthesis or secretion of proOmpA in trigger factor-depleted cells. The primary physiological defect in trigger factor-depleted or -overproducing cells is an enrichment of filamented cells. Filamentation of the trigger factor-overproducing strain is suppressed by a multicopy plasmid expressing the essential division gene ftsZ, suggesting that trigger factor has an important role in cell division.     

Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes.

The results from experiments involving nonradiative energy transfer indicate that a fluorescent probe on the 5'-end of tRNA(Phe) moves more than 20 A towards probes on ribosomal protein L1 as a peptide bond is formed during the peptidyl transferase reaction on Escherichia coli ribosomes. The peptide itself moves no more than a few angstroms during peptide bond formation, as judged by the movement of fluorescent probes attached to the phenylalanine amino group of phenylalanyl-tRNA. Other results demonstrate that an analogue of peptidyl-tRNA, deacylated tRNA, and puromycin can be bound simultaneously to the same ribosome, indicating that there are three physically distinct sites to which tRNA is bound during the reaction steps by which peptides are elongated. The results appear to be consistent with the displacement model of peptide elongation.     

Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction

The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. We report how endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Our studies show that Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and that both Hh and Ptc are targeted to lysosomal degradation. We also found that the ptc(14) mutant does not block Hh spreading, as it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, we noted that both in this mutant and in those not producing Ptc protein, Hh still occurred in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc-independent, mechanism of Hh internalization.     

Trigger factor binding to ribosomes with nascent peptide chains of varying lengths and sequences

Trigger factor (TF) is the first protein-folding chaperone to interact with a nascent peptide chain as it emerges from the ribosome. Here, we have used a spin down assay to estimate the affinities for the binding of TF to ribosome nascent chain complexes (RNCs) with peptides of varying lengths and sequences. An in vitro system for protein synthesis assembled from purified Escherichia coli components was used to produce RNCs stalled on truncated mRNAs. The affinity of TF to RNCs exposing RNA polymerase sequences increased with the length of the nascent peptides. TF bound to RNA polymerase RNCs with significantly higher affinity than to inner membrane protein leader peptidase and bacterioopsin RNCs. The latter two RNCs are substrates for signal recognition particle, suggesting complementary affinities of TF and signal recognition particle to nascent peptides targeted for cytoplasm and membrane.     

HtrA2 interacts with A beta peptide but does not directly alter its production or degradation

HtrA2/Omi is a mammalian mitochondrial serine protease homologous to the E. coli HtrA/DegP gene products. Recently, HtrA2/Omi was found to have a dual role in mammalian cells, acting as an apoptosis-inducing protein and being involved in maintenance of mitochondrial homeostasis. By screening a human brain cDNA library with A beta peptide as bait in a yeast two-hybrid system, we identified HtrA2/Omi as a binding partner of A beta peptide. The interaction between A beta peptide and HtrA2/Omi was confirmed by an immunoblot binding assay. The possible involvement of HtrA2/Omi in A beta peptide metabolism was investigated. In vitro peptide cleavage assays showed that HtrA2/Omi did not directly promote the production of A beta peptide at the beta/gamma-secretase level, or the degradation of A beta peptide. However, overexpression of HtrA2/Omi in K269 cells decreased the production of A beta40 and A beta42 by up to 30%. These results rule out the involvement of HtrA2/Omi in the etiology of Alzheimer's disease. However, the fact that overexpression of HtrA2/Omi reduces the generation of A beta40 and A beta42 suggests that it may play some positive role in mammalian cells.     

Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells

Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.     

Mutational alterations affecting the export competence of a truncated but fully functional maltose-binding protein signal peptide.

The wild-type maltose-binding protein (MBP) signal peptide is 26 amino acids in length. A mutationally altered MBP signal peptide has been previously described that is missing one of the basic residues from the hydrophilic segment and seven residues from the hydrophobic core; however, it still facilitates MBP secretion to the periplasm at a rate and efficiency comparable to those of the wild-type structure. Thus, this truncated signal peptide (designated the R2 signal peptide) must retain all of the essential features required for proper export function. In this study, alterations were obtained in the R2 signal peptide that resulted in an export-defective MBP. For the first time, signal sequence mutations were obtained that resulted in the synthesis of a totally export-defective MBP. As was previously the case for the wild-type signal peptide, the introduction of either charged residues or helix-breaking proline residues adversely affected export function. Despite these similarities, the position of these alterations within the R2 signal peptide, their relative effects on MBP secretion and processing, and an analysis of the ability of various extragenic prl mutations to suppress the secretion defects provide additional insight into the minimal requirements for a functional MBP signal peptide.     

Trigger factor assists the refolding of heterodimeric but not monomeric luciferases

The refolding of thermally inactivated protein by ATP-independent trigger factor (TF) and ATP-dependent DnaKJE chaperones was comparatively analyzed. Heterodimeric (αβ) bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi as well as monomeric luciferases of Vibrio harveyi and Luciola mingrelica (firefly) were used as substrates. In the presence of TF, thermally inactivated heterodimeric bacterial luciferases refold, while monomeric luciferases do not refold. These observations were made both in vivo (Escherichia coli ΔdnaKJ containing plasmids with tig gene) and in vitro (purified TF). Unlike TF, the DnaKJE chaperone system refolds both monomeric and heterodimeric luciferases with equal efficiency.     

h-ANF does not play a role in the regulation of myocardial force of contraction

In the present study, h-ANF failed to reduce myocardial force of contraction alone and in the presence of isoprenaline. This lack of effect was observed in electrically driven, isolated human papillary muscle strips and rat papillary muscles. In contrast, adenosine and carbachol exerted antiadrenergic actions in rat and human myocardium. It is concluded that h-ANF obviously does not play a role in the regulation of human and rat myocardial force of contraction, whereas adenosine and carbachol are capable to produce antiadrenergic effects in the human and rat myocardium.     

Mitochondrial alternative oxidase is not a critical component of plant viral resistance but may play a role in the hypersensitive response

Transgenic tobacco (Nicotiana tabacum) with altered levels of mitochondrial alternative oxidase (AOX) were used to examine the potential role of this electron transport chain protein in resistance to tobacco mosaic virus. We examined the effect of AOX expression on the salicylic acid-induced resistance in susceptible plants and the resistance responses of plants harboring the N-gene. A lack of AOX did not compromise the ability of salicylic acid treatment to heighten the resistance of susceptible plants. In plants with the N-gene, a lack of AOX did not compromise the ability of the hypersensitive response to restrict the virus or the ability of the plant to develop systemic acquired resistance. Overexpression of AOX did not heighten the resistance of susceptible plants, but did result in smaller hypersensitive response lesions, suggesting a link between mitochondrial function and this programmed cell death event. We conclude that AOX is not a critical component of the previously characterized salicylhydroxamic acid-sensitive pathway important in viral resistance.     

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of PprI’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. ra-diodurans R1 strain, pprI function-deficient mutant (YR1), and pprI function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence mi-croscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells ex-hibit the tightly packed ring-like morphology, while the pprI function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprI completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprI partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a PprI C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensi-tive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.     

Soybean ureide transporters play a critical role in nodule development,function and nitrogen export

Legumes can access atmospheric nitrogen through a symbiotic relationship with nitrogen‐fixing bacteroids that reside in root nodules. In soybean, the products of fixation are the ureides allantoin and allantoic acid, which are also the dominant long‐distance transport forms of nitrogen from nodules to the shoot. Movement of nitrogen assimilates out of the nodules occurs via the nodule vasculature; however, the molecular mechanisms for ureide export and the importance of nitrogen transport processes for nodule physiology have not been resolved. Here, we demonstrate the function of two soybean proteins – GmUPS1‐1 (XP_003516366) and GmUPS1‐2 (XP_003518768) – in allantoin and allantoic acid transport out of the nodule. Localization studies revealed the presence of both transporters in the plasma membrane, and expression in nodule cortex cells and vascular endodermis. Functional analysis in soybean showed that repression of GmUPS1‐1 and GmUPS1‐2 in nodules leads to an accumulation of ureides and decreased nitrogen partitioning to roots and shoot. It was further demonstrated that nodule development, nitrogen fixation and nodule metabolism were negatively affected in RNAi UPS1 plants. Together, we conclude that export of ureides from nodules is mediated by UPS1 proteins, and that activity of the transporters is not only essential for shoot nitrogen supply but also for nodule development and function.     

CD36 does not play a direct role in HDL or LDL metabolism

CD36 and scavenger receptor class B, type I (SR-BI) are both class B scavenger receptors that recognize a broad variety of ligands, including oxidized low density lipoprotein (oxLDL), HDL, anionic phospholipids, and apoptotic cells. In this study we investigated the role of mouse CD36 (mCD36) as a physiological lipoprotein receptor. We compared the association of various lipoprotein particles with mCD36 and mSR-BI expressed in COS cells by adenovirus-mediated gene transfer. mCD36 bound human oxLDL and mouse HDL with high affinity. Human LDL bound poorly to mCD36, indicating that mCD36 is unlikely to play a significant role in LDL metabolism. The ability of mCD36 to mediate the selective uptake of cholesteryl esters (CE) from receptor-bound HDL was assessed. In comparison with mSR-BI, mCD36 inefficiently mediated the selective uptake of CE. Hepatic overexpression of mCD36 in C57BL/6 mice by adenovirus-mediated gene transfer did not result in significant alterations in plasma LDL and HDL levels. We conclude that mCD36, while able to bind HDL with high affinity, does not contribute significantly to HDL or LDL metabolism.     

The Sp1 transcription factor does not directly interact with the HIV-1 Tat protein

The role of Sp1 in regulating the trans-activating activity of the human immunodeficiency virus type 1 (HIV-1) Tat protein has not yet been clearly defined. In fact, studies on the physical and functional interaction between Sp1 and Tat have yielded contradictory results. Here we investigated whether a physical interaction between Sp1 and Tat indeed occurs, exploiting both biochemical and genetic techniques that allow detection of direct protein-protein interactions. Studies performed with the yeast two-hybrid system indicate that Sp1 does not directly interact with the HIV-1 Tat protein. Control experiments demonstrated that both proteins are functionally expressed in the yeast cells. In vitro binding assays further confirmed that Sp1 does not physically bind Tat. These data suggest that in vivo Tat and Sp1 most likely take part of a multicomponent complex and thus encourage the search of the molecule(s) which mediate Tat-Sp1 interaction.     

Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products.     

DmsD is required for the biogenesis of DMSO reductase in Escherichia coli but not for the interaction of the DmsA signal peptide with the Tat apparatus

The DmsD protein is essential for the biogenesis of DMSO reductase in Escherichia coli, and binds the signal peptide of the DmsA subunit, a Tat substrate. This suggests a role as a guidance factor to target pre-DmsA to the translocase. Here, we have analysed the export of fusion proteins in which the DmsA and TorA signal peptides are fused to green fluorescent protein. Both chimeras are efficiently exported to the periplasm in wild-type E. coli cells and we show that their export efficiencies are essentially identical in a mutant lacking DmsD. An authentic Tat substrate, TMAO reductase, is also efficiently exported in the dmsD mutant. The data indicate that DmsD carries out a critical role in DMSO reductase biogenesis/assembly but is not required for the functioning of the DmsA signal peptide.     

A genetic screen for suppressors of Escherichia coli Tat signal peptide mutations establishes a critical role for the second arginine within the twin-arginine motif

The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.     

EGF-like peptide of Dictyostelium discoideum is not a chemoattractant but it does restore folate-mediated chemotaxis in the presence of signal transduction inhibitors

A synthetic EGF-like (EGFL) peptide (DdEGFL1), equivalent to the first EGFL domain in the extracellular matrix protein CyrA, has previously been shown to enhance random cell motility and cAMP-mediated chemotaxis in Dictyostelium discoideum. However the role of DdEGFL1 as a potential chemoattractant had not been addressed. In this study, a micropipette assay and an under-agarose migration assay showed that DdEGFL1 is not a chemoattractant for Dictyostelium cells. A radial bioassay was used to show that DdEGFL1 does not significantly enhance folate-mediated chemotaxis in contrast to its chemokinetic effect during chemotaxis toward cAMP. However, DdEGFL1 was able to rescue chemotaxis toward folate when the pathway was inhibited by pharmacological agents that inhibit known components of the signaling cascade (e.g. phosphatidylinositol 3-kinase, phospholipase A2, tyrosine kinases, and calmodulin). These data suggest that DdEGFL1 may activate a novel motility pathway that when coupled with folic acid receptor activation, can maintain the normal migratory response to folic acid in vegetative cells. Together, this data provides new insight into the function of EGFL repeats during Dictyostelium chemotaxis and the existence of a novel motility pathway regulated by EGFL peptides and/or repeats in this model organism.