PF3 encodes a DRC subunit required for assembly of the DRC-nexin link
Maureen Wirschell/b>1, Douglas Tritschler2, Raqual Bower2, Winfield Sale1, and Mary E. Porter2
1) Emory University, Atlanta, GA, USA
2) University of Minnesota, Minneapolis, MN, USA
The dynein regulatory complex (DRC) is a group of tightly bound axonemal polypeptides (DRC1-DRC7) located at the junction between radial spoke 2 and the dynein arms (Piperno et al., 1994; Gardner et al., 1994). Drc mutations restore motility to paralyzed central pair (CP)/radial spoke (RS) mutants leading to the hypothesis that the DRC plays a key role in mediating signals between the RS and dyneins arms (Huang et al., 1982). CryoET has shown that the DRC is part of the nexin link that interconnects outer doublets and is thought to limit microtubule sliding during bending (Heuser et al., 2009). The DRC-nexin link has a complex sub-structure and is much larger than previously realized. Here, we report the identification of DRC1 as the PF3 gene product. Direct amino acid sequencing identified two peptides with sequence homology to a conserved protein in Volvox and humans. However, only small fragments of the sequence are found in the Chlamydomonas genome. We therefore used PCR-based mapping to confirm linkage to the pf3 mutation, and RT-PCR and 5'RACE to recover a 2520-bp cDNA sequence encoding the full-length DRC1 polypeptide. The wild-type DRC1 sequence contains 698 amino acids with a predicted molecular weight of 79.3 kD. The pf3-1 mutation is C to A transition resulting in a premature stop codon after the first five amino acids. Affinity-purified antibodies confirm that DRC1 is found at wild-type levels in pf2, sup-pf-3 and sup-pf-4 axonemes, but is missing in pf3. However, PF3/DRC1 co-extracts and co-IPs with PF2/DRC4. These observations are consistent with the hypothesis that DRC1 and DRC4 are DRC subunits that are located in distinct sub-domains of the DRC-nexin link.
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