Cytoplasmic assembly of the flagellar inner arm dynein I1 / f dynein and characterization of ida3
Rasagnya Viswanadha1, Maureen Wirschell1, Laura A. Fox1, Tal Kramer2, Masafumi Hirono3, Ritsu Kamiya3, and Winfield S. Sale1
1Cell Biology, Emory University School of Medicine, Atlanta, GA 30322 USA; 2Molecular Biology, Princeton University, New Jersey, 3Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
Our goal is to understand the mechanism of assembly of the inner dynein arm, I1/ f-dynein, a conserved axonemal dynein required for normal flagellar motility. I1 dynein sediments as a large, 20S complex composed of two heavy chains, three intermediate chains and several light chains, and is located at the proximal end of the 96-nm axonemal repeat. Biochemical analysis of wild-type cytoplasmic extracts revealed that I1-dynein can assemble into a 20S complex in the cytoplasm before being transported to the flagellar compartment. To further determine how I1 dynein is assembled, we have taken advantage of the Chlamydomonas mutant ida3 that fails to assemble I1 dynein in the axoneme (Kamiya et al., 1991. JCB 112: 441-447). The ida3 mutant maps to LG III and IDA3 does not encode any of the known I1-dynein proteins. Thus, we predicted that IDA3 encodes a protein, Ida3p, required for: [a] cytoplasmic assembly of I1 dynein; [b] interaction of I1 dynein with IFT; or [c] docking of I1 dynein in the axoneme. Biochemical fractionation of ida3 cytoplasmic extracts revealed that I1 dynein can assemble into 20S complexes as in wild-type cells. Moreover, in vitro reconstitution analysis revealed that I1 dynein from axonemal extracts can rebind dynein-depleted axonemes from wild-type and ida3 cells. These results suggest that the ida3 mutant is neither deficient in a protein required for cytoplasmic assembly of the 20S I1-dynein complex nor for a docking protein required for anchoring I1 dynein in the axoneme. Thus, we postulate that Ida3p is required for interaction between I1 dynein and the IFT machinery for transport to the flagellar compartment. To test this idea, we will clone IDA3 and characterize the Ida3 protein.
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