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Essential Determinant (essential + determinant)
Selected AbstractsThe carboxy-terminal tail region of human Cav2.1 (P/Q-type) channel is not an essential determinant for its subcellular localization in cultured neuronesGENES TO CELLS, Issue 2 2005Qiuping Hu A recent report on the mechanism of synaptic targeting of Cav2.2 channel suggested that this process depends upon the presence of long C-terminal tail and that protein interactions mediated by SH3-binding and PDZ-binding motifs in the tail region are important. To examine the possibility that C-terminal tail of the Cav2.1 channel and the polyglutamine stretch therein are also involved in the mechanism for channel localization, we constructed several expression plasmids for human Cav2.1 channel tagged with enhanced green fluorescent protein (EGFP) and introduced them into mouse hippocampal neuronal culture. HC construct encodes short version of Cav2.1, and HS and HL encode Cav2.1 channel with a long C-terminal tail, which contains polyglutamine tract of 13 (normal range) and 28 (SCA6 disease range) repeat units, respectively. Surprisingly, transfection with HC, HS, and HL gave essentially the same results: EGFP signal was observed in cell soma, dendrites, and the axon as well. Furthermore, mutation of the PDZ-binding motif located at the C-terminus of the long version of Cav2.1, by adding FLAG tag, did not affect the localization patterns of HS and HL as well. Therefore, the C-terminal region is not indispensable for the subcellular localization of Cav2.1 channel, nor expansion of polyglutamine length affected the localization of the channel. Thus, it is possible that the localization mechanism of Cav2.1 channel is different from that of Cav2.2, though these channels share various structural and functional characteristics. [source] Interaction between the RP4 coupling protein TraG and the pBHR1 mobilization protein MobMOLECULAR MICROBIOLOGY, Issue 6 2000Cédric Y. Szpirer It is currently believed that interaction between the relaxosome of a mobilizable plasmid and the transfer machinery of the helper conjugative plasmid is mediated by a TraG family coupling protein. The coupling proteins appear as an essential determinant of mobilization specificity and efficiency. Using a two-hybrid system, we demonstrated for the first time the direct in vivo interaction between the coupling protein of a conjugative plasmid (the TraG protein of RP4) and the relaxase of a mobilizable plasmid (the Mob protein of pBHR1, a derivative of the broad host range plasmid pBBR1). This interaction was confirmed in vitro by an overlay assay and was shown to occur even in the absence of the transfer origin of pBHR1. We showed that, among 11 conjugative plasmids tested, pBHR1 is efficiently mobilized only by plasmids encoding an IncP-type transfer system. We also showed that the RP4 TraG coupling protein is essential for mobilization of a pBBR1 derivative and is the element that allows its mobilization by R388 plasmid (IncW) at a detectable frequency. [source] Non-invasive quantification of endogenous root auxin transport using an integrated flux microsensor techniqueTHE PLANT JOURNAL, Issue 6 2010Eric S. McLamore Summary Indole-3-acetic acid (IAA) is a primary phytohormone that regulates multiple aspects of plant development. Because polar transport of IAA is an essential determinant of organogenesis and dynamic tropic growth, methods to monitor IAA movement in vivo are in demand. A self-referencing electrochemical microsensor was optimized to non-invasively measure endogenous IAA flux near the surface of Zea mays roots without the addition of exogenous IAA. Enhanced sensor surface modification, decoupling of acquired signals, and integrated flux analyses were combined to provide direct, real time quantification of endogenous IAA movement in B73 maize inbred and brachytic2 (br2) auxin transport mutant roots. BR2 is localized in epidermal and hypodermal tissues at the root apex. br2 roots exhibit reduced shootward IAA transport at the root apex in radiotracer experiments and reduced gravitropic growth. IAA flux data indicates that maximal transport occurs in the distal elongation zone of maize roots, and net transport in/out of br2 roots was decreased compared to B73. Integration of short term real time flux data in this zone revealed oscillatory patterns, with B73 exhibiting shorter oscillatory periods and greater amplitude than br2. IAA efflux and influx were inhibited using 1- N -naphthylphthalamic acid (NPA), and 2-naphthoxyacetic acid (NOA), respectively. A simple harmonic oscillation model of these data produced a correlation between modeled and measured values of 0.70 for B73 and 0.69 for br2. These results indicate that this technique is useful for real-time IAA transport monitoring in surface tissues and that this approach can be performed simultaneously with current live imaging techniques. [source] SlCCD7 controls strigolactone biosynthesis, shoot branching and mycorrhiza-induced apocarotenoid formation in tomatoTHE PLANT JOURNAL, Issue 2 2010Jonathan T. Vogel Summary The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch-inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C13 cyclohexenone and C14 mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source,sink interactions and production of arbuscular mycorrhiza-induced apocarotenoids. [source] The PDZ domain protein CAL interacts with mGluR5a and modulates receptor expressionJOURNAL OF NEUROCHEMISTRY, Issue 3 2010Shan Cheng J. Neurochem. (2010) 112, 588,598. Abstract In this study, we investigated the association of metabotropic glutamate receptor subtype-5a (mGluR5a) with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL). Using glutathione- S -transferase pull-down techniques, we found that mGluR5a directly interacted with CAL, with the C-terminus of the receptor binding to the PSD95/Discslarge/ZO-1 homology domain of CAL. The last four amino acids (S-S-S-L) of the C-terminus of the receptor were essential determinants for the interaction. Co-immunoprecipitation experiments and immunofluorescence assays revealed that full-length mGluR5a also associated with intact CAL in vivo, an observation consistent with the results from studies on fragment interactions in vitro. Functionally, upon co-expression with mGluR5a, CAL profoundly inhibited the ubiquitination of mGluR5a and enhanced receptor expression at the protein level but not at the mRNA level. These findings reveal that mGluR5a protein expression is physiologically regulated via its interaction with CAL. These results also suggest a molecular mechanism by which mGluR5a protein expression may be regulated at the post-translational level by the CAL protein, possibly by blocking ubiquitination-dependent receptor degradation. [source] A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor functionTHE JOURNAL OF PHYSIOLOGY, Issue 4 2010Dai-Fei Wu The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT3) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the ,-aminobutyric acid type A (GABAA) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT3A receptors, we replaced it with alanine and expressed the 5-HT3A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT3A(C312A) mutant, when co-expressed with the wild-type 5-HT3A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT3A(C312A) with 5-HT3B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT3A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor , and , subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor ,7 and GABAA, subunits) supported robust function. Thus, 5-HT3A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating. [source] | ||||||||||