Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits

Ben F Lucker; Robert H Behal; Hongmin Qin; Laura C Siron; W David Taggart; Joel L Rosenbaum; Douglas G Cole

doi:10.1074/jbc.M505062200

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Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits

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Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits

Ben F Lucker, Robert H Behal, Hongmin Qin, Laura C Siron, W David Taggart, Joel L Rosenbaum and Douglas G Cole

The Journal of biological chemistry, Vol.280(30), pp.27688-27696

07/29/2005

DOI: https://doi.org/10.1074/jbc.M505062200

Handle:

https://hdl.handle.net/2376/111075

PMID: 15955805

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Abstract

Carrier Proteins - physiology

Protein Structure, Tertiary

Temperature

Cross-Linking Reagents - pharmacology

Cytoplasm - metabolism

Intracellular Signaling Peptides and Proteins - metabolism

Two-Hybrid System Techniques

Blotting, Northern

Animals

Carrier Proteins - metabolism

Flagella - metabolism

Trypsin - pharmacology

Membrane Proteins - physiology

Models, Biological

Cloning, Molecular

Protein Binding

Chlamydomonas reinhardtii - metabolism

Membrane Proteins - metabolism

Models, Genetic

Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

DNA, Complementary - metabolism

Hydrogen-Ion Concentration

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.

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Title: Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits
Creators: Ben F Lucker - Department of Microbiology, Molecular Biology, and Biochemistry and the Center for Reproductive Biology, LSS142, University of Idaho, Moscow, Idaho 83844, USA
Robert H Behal
Hongmin Qin
Laura C Siron
W David Taggart
Joel L Rosenbaum
Douglas G Cole
Publication Details: The Journal of biological chemistry, Vol.280(30), pp.27688-27696
Academic Unit: Center for Reproductive Biology
Publisher: United States
Grant note: P20RR16454 / NCRR NIH HHS GM14642 / NIGMS NIH HHS GM61920 / NIGMS NIH HHS
Identifiers: 99900547285401842
Language: English
Resource Type: Journal article