Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits.
Required for the assembly and maintenance of eukaryotic cilia and flagella, intraflagellar transport (IFT) consists of the bidirectional movement of large protein particles between the base and the distal tip of the organelle. Anterograde movement of particles away from the cell body is mediated by kinesin-2, whereas retrograde movement away from the flagellar tip is powered by cytoplasmic dynein 1b/2. IFT particles contain multiple copies of two distinct protein complexes, A and B, which contain at least 6 and 11 protein subunits, respectively. In this study, we have used increased ionic strength to remove four peripheral subunits from the IFT complex B of Chlamydomonas reinhardtii, revealing a 500-kDa core that contains IFT88, IFT81, IFT74/72, IFT52, IFT46, and IFT27. This result demonstrates that the complex B subunits, IFT172, IFT80, IFT57, and IFT20 are not required for the core subunits to stay associated. Chemical cross-linking of the complex B core resulted in multiple IFT81-74/72 products. Yeast-based two-hybrid and three-hybrid analyses were then used to show that IFT81 and IFT74/72 directly interact to form a higher order oligomer consistent with a tetrameric complex. Similar analysis of the vertebrate IFT81 and IFT74/72 homologues revealed that this interaction has been evolutionarily conserved. We hypothesize that these proteins form a tetrameric complex, (IFT81)2(IFT74/72)2, which serves as a scaffold for the formation of the intact IFT complex B.
Lucker
Ben F
BF
Department of Microbiology, Molecular Biology, and Biochemistry and the Center for Reproductive Biology, LSS142, University of Idaho, Moscow, Idaho 83844, USA.
Behal
Robert H
RH
Qin
Hongmin
H
Siron
Laura C
LC
Taggart
W David
WD
Rosenbaum
Joel L
JL
Cole
Douglas G
DG
D000818:Animals; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D016825:Chlamydomonas reinhardtii / Q000378:metabolism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005407:Flagella / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D008957:Models, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D014357:Trypsin