@article{12082, abstract = {Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.}, author = {Artan, Murat and Hartl, Markus and Chen, Weiqiang and De Bono, Mario}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {9}, publisher = {Elsevier}, title = {{Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans}}, doi = {10.1016/j.jbc.2022.102343}, volume = {298}, year = {2022}, } @article{10117, abstract = {Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.}, author = {Artan, Murat and Barratt, Stephen and Flynn, Sean M. and Begum, Farida and Skehel, Mark and Nicolas, Armel and De Bono, Mario}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {3}, publisher = {Elsevier}, title = {{Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling}}, doi = {10.1016/J.JBC.2021.101094}, volume = {297}, year = {2021}, } @article{4179, abstract = {Neurotrophin-3 (NT-3) is a member of the neurotrophin gene family and is highly expressed in the developing rat cerebellum. Here we show that brain-derived neurotrophic factor (BDNF) increased by approximately 10-fold the NT-3 mRNA levels in cultured cerebellar granule neurons isolated from postnatal rats, whereas nerve growth factor (NGF) and NT-3 itself had no effect. The effect of BDNF was additive to that of triiodothyronine (T3), which also increased NT-3 mRNA in these neurons. The drug K252a inhibited the BDNF-mediated stimulation of NT-3 expression, suggesting an involvement of trkB receptors. Nuclear run-on experiments showed that BDNF enhanced NT-3 transcription, whereas the stability of NT-3 mRNA remained unchanged. The data presented are the first demonstration that one neurotrophin regulates the expression of another and provide evidence that NT-3 production in granule neurons is regulated by both BDNF and T3.}, author = {Leingärtner, Axel and Heisenberg, Carl-Philipp J and Kolbeck, Roland and Thoenen, Hans and Lindholm, Dan}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {2}, pages = {828 -- 830}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Brain-derived neurotrophic factor increases neurotrophin-3 expression in cerebellar granule neurons}}, doi = {10.1016/s0021-9258(17)42186-7}, volume = {269}, year = {1994}, } @article{2480, abstract = {Functional cDNA clones for rat neuromedin K receptor were isolated from a rat brain cDNA library by cross-hybridization with the bovine substance K recepor cDNA. Injection of the mRNA synthesized in vitro from the cloned cDNA into Xenopus oocytes elicited electrophysiological responses to tachykinins, with the most potent sensitivity being to neuromedin K. Ligand-binding displacement in membranes of mammalian COS cells transfected with the cDNA indicated the rank order of affinity of the receptor to tachykinins; neuromedin K > substance K > substance P. The hybridization analysis showed that the neuromedin K receptor mRNA is expressed in both the brain and the peripheral tissues at different levels. The rat neuromedin K receptor consists of 452 amino acid residues and belongs to the family of G protein-coupled receptors, which are thought to have seven transmembrane domains. The sequence comparison of the rat neuromedin K, substance P, and substance K receptors revealed that these receptors are highly conserved in the seven transmembrane domains and the cytoplasmic sides of the receptors. They also show some structural characteristics, including the common presence of histidine residues in transmembrane segments V and VI and the difference in the numbers and distributions of serine and threonine residues as possible phosphorylation sites in the cytoplasmic regions. This paper thus presents the first comprehensive analysis of the molecular nature of the multiple peptide receptors that exhibit similar but pharmacologically distinguishable activities.}, author = {Shigemoto, Ryuichi and Yokota, Yoshifumi and Tsuchida, Kunihiro and Nakanishi, Shigetada}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {2}, pages = {623 -- 628}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Cloning and expression of a rat neuromedin K receptor cDNA}}, doi = {10.1016/s0021-9258(19)40095-1 }, volume = {265}, year = {1990}, } @article{2525, abstract = {This paper describes the amino acid sequence of the rat substance P receptor and its comparison with that of the rat substance K receptor on the basis of molecular cloning and sequence analysis. From a rat brain cDNA library constructed with an RNA expression vector, we identified a cDNA mixture containing a functional substance P receptor cDNA by examining electrophysiologically a receptor expression following injection of the mRNAs synthesized in vitro into Xenopus oocytes. A receptor cDNA clone was then isolated by cross-hybridization with the bovine substance K receptor DNA. The clone was confirmed by selective binding of substance P to the cloned receptor expressed in mammalian COS cells. The deduced amino acid sequence (407 amino acid residues) possesses seven putative membrane spanning domains and shows a sequence similarity to the members of G-protein-coupled receptors. The rat substance P and substance K receptor are very similar in both size and amino acid sequences, particularly in the putative transmembrane similarity is in marked contrast to the sequence divergence in the amino- and carboxyl-terminal regions and the third cytoplasmic loop. The observed sequence similarytity and divergence would thus contribute to the expression of similar but pharmacological regions and the first and second cytoplasmic loops. This distinguishable activities of the two tachykinin receptors.}, author = {Yokota, Yoshifumi and Sasai, Yoshiki and Tanaka, Kohichi and Fujiwara, Tsutomu and Tsuchida, Kunihiro and Shigemoto, Ryuichi and Kakizuka, Akira and Ohkubo, Hiroaki and Nakanishi, Shigetada}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {30}, pages = {17649 -- 17652}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Molecular characterization of a functional cDNA for rat substance P receptor}}, doi = {doi.org/10.1016/S0021-9258(19)84619-7}, volume = {264}, year = {1989}, }