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      ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

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          Abstract

          Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

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          Neurotransmitter synthesis and uptake by isolated sympathetic neurones in microcultures.

          Assays of isolated single sympathetic neurones show that their transmitter functions can be either adrenergic or cholinergic depending on growth conditions. The data suggest that the number of transmitters made by most mature individual neurones is restricted.
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            Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum.

            The capacity of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer can be largely abolished by extracting them with high ionic strength buffers. It can be reconstituted by adding the salt extract back to the depleted membranes [Warren, G. & Doberstein, B. (1978) Nature (London) 273, 569-571]. Utilizing hydrophobic chromatography, we purified to homogeneity a protein component of the salt extract that reconstitutes the translocation activity of the extracted membranes. This component behaves as a homogeneous species upon gel filtration, ion-exchange chromatography, adsorption chromatography, and sucrose-gradient centrifugation. When examined by polyacrylamide gel electrophoresis in NaDodSO4, six polypeptides with apparent Mr of 72,000, 68,000, 54,000, 19,000, 14,000, and 9000 are observed in about equal and constant stoichiometry, suggesting that they are subunits of a complex. The sedimentation coefficient of 11S is in good agreement with the sum of the Mr of the subunits. The Mr 68,000 and 9000 subunits label intensely with N-[3H]ethylmaleimide. Thus, the reported sulfhydryl group requirement of the translocation activity in the unfractionated extract [Jackson, R. C., Walter, P. & Blobel, G. (1980) Nature (London), 286, 174-176] may be localized to either or both the Mr 68,000 and 9000 subunits of the purified complex.
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              Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene.

              Cilia and flagella are microtubule-based structures nucleated by modified centrioles termed basal bodies. These biochemically complex organelles have more than 250 and 150 polypeptides, respectively. To identify the proteins involved in ciliary and basal body biogenesis and function, we undertook a comparative genomics approach that subtracted the nonflagellated proteome of Arabidopsis from the shared proteome of the ciliated/flagellated organisms Chlamydomonas and human. We identified 688 genes that are present exclusively in organisms with flagella and basal bodies and validated these data through a series of in silico, in vitro, and in vivo studies. We then applied this resource to the study of human ciliation disorders and have identified BBS5, a novel gene for Bardet-Biedl syndrome. We show that this novel protein localizes to basal bodies in mouse and C. elegans, is under the regulatory control of daf-19, and is necessary for the generation of both cilia and flagella.
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                Author and article information

                Journal
                J Cell Biol
                jcb
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                20 October 2008
                : 183
                : 2
                : 313-322
                Affiliations
                [1 ]Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY 13210
                [2 ]Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844
                Author notes

                Correspondence to David R. Mitchell: mitcheld@ 123456upstate.edu

                Article
                200802025
                10.1083/jcb.200802025
                2568026
                18852297
                ed757bc1-e777-40f5-b909-bdfccdb6190e
                © 2008 Ahmed et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

                History
                : 4 February 2008
                : 10 September 2008
                Categories
                Research Articles
                Article

                Cell biology
                Cell biology

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