The approximately 20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains approximately 16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction.
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http://dx.doi.org/10.1073/pnas.0500553102 | DOI Listing |
Methods Mol Biol
January 2025
Institute of Neurophysiology and NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Berlin, Germany.
The bimolecular fluorescence complementation (BiFC) technique is a powerful tool for visualizing protein-protein interactions in vivo. It involves genetically fused nonfluorescent fragments of green fluorescent protein (GFP) or its variants to the target proteins of interest. When these proteins interact, the GFP fragments come together, resulting in the reconstitution of a functional fluorescent protein complex that can be observed using fluorescence microscopy.
View Article and Find Full Text PDFThe glycoprotein hormones of humans, produced in the pituitary and acting through receptors in the gonads to support reproduction and in the thyroid gland for metabolism, have co-evolved from invertebrate counterparts . These hormones are heterodimeric cystine-knot proteins; and their receptors bind the cognate hormone at an extracellular domain and transmit the signal of this binding through a transmembrane domain that interacts with a heterotrimeric G protein. Structures determined for the human receptors as isolated for cryogenic electron microscopy (cryo-EM) are all monomeric despite compelling evidence for their functioning as dimers .
View Article and Find Full Text PDFCannulae are tubular protein filaments that accumulate on the extracellular surface of the hyperthermophilic archaeon during cell division. Cannulae have been postulated to act as a primitive extracellular matrix through which cells could communicate or exchange material, although their native biological function remains obscure. Here, we report cryoEM structural analyses of cannulae and of protein assemblies derived from recombinant cannula-like proteins.
View Article and Find Full Text PDFPlant Cell Rep
January 2025
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China.
An endoplasmic reticulum-localized Cu transporter, PhHMA5II1, interacts with copper chaperones and plays an important role in Cu detoxification in petunia. Copper (Cu) is an essential element for plant growth but toxic when present in excess. In this study we present the functional characterization of a petunia (Petunia hybrida) P-type heavy-metal ATPases (HMAs), PhHMA5II1.
View Article and Find Full Text PDFBiochem Biophys Res Commun
January 2025
Department of Bioscience, Tokyo University of Agriculture, Tokyo, 156-8502, Japan. Electronic address:
Plant responses to the water environment are mediated by ethylene (submergence response) and abscisic acid (ABA, drought response). Ethylene is perceived by a family of histidine kinase receptors (ETR-HKs), which regulate the activity of the downstream B3 Raf-like (RAF) kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) in an ethylene-dependent manner. We previously demonstrated in the moss Physcomitrium patens that SNF1-related protein kinase 2 (SnRK2), an essential kinase in osmostress responses in land plants, is activated by the B3-RAF kinase ARK, which is also regulated by ETR-HKs in an ABA- and osmostress-dependent manner.
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