title: Subunit interactions and organization of the Chlamydomonas reinhardtii intraflagellar transport complex A proteins.
abstract: Chlamydomonas reinhardtii intraflagellar transport (IFT) particles can be biochemically resolved into two smaller assemblies, complexes A and B, that contain up to six and 15 protein subunits, respectively. We provide here the proteomic and immunological analyses that verify the identity of all six Chlamydomonas A proteins. Using sucrose density gradient centrifugation and antibody pulldowns, we show that all six A subunits are associated in a 16 S complex in both the cell bodies and flagella. A significant fraction of the cell body IFT43, however, exhibits a much slower sedimentation of ∼2 S and is not associated with the IFT A complex. To identify interactions between the six A proteins, we combined exhaustive yeast-based two-hybrid analysis, heterologous recombinant protein expression in Escherichia coli, and analysis of the newly identified complex A mutants, ift121 and ift122. We show that IFT121 and IFT43 interact directly and provide evidence for additional interactions between IFT121 and IFT139, IFT121 and IFT122, IFT140 and IFT122, and IFT140 and IFT144. The mutant analysis further allows us to propose that a subset of complex A proteins, IFT144/140/122, can form a stable 12 S subcomplex that we refer to as the IFT A core. Based on these results, we propose a model for the spatial arrangement of the six IFT A components.
authors.affiliation: Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, Idaho 83844, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Behal
authors.forename: Mark S
authors.initials: MS
authors.lastname: Miller
authors.forename: Hongmin
authors.initials: H
authors.lastname: Qin
authors.forename: Ben F
authors.initials: BF
authors.lastname: Lucker
authors.forename: Alexis
authors.initials: A
authors.lastname: Jones
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005407:Flagella / Q000737:chemistry / Q000378:metabolism*; D055786:Gene Knockout Techniques; D047468:Immunoprecipitation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D046912:Multiprotein Complexes; D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins