The Chlamydomonas paralyzed flagellar mutant pf4 is defective in the PP2A B-subunit and reveals that PP2A is required for normal motility and phototaxis

Candice A. Elam1, Maureen Wirschell1, Laura A. Fox1, Kerry York2, Susan K. Dutcher2, and Winfield S. Sale1

1) Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA 2) Genetics, Washington University School of Medicine, St. Louis, MO 63110 USA

The overall goal of the work is to determine the mechanisms that regulate dynein and flagellar motility. Our previous studies revealed that the PP2A A- and C-subunits are localized to the outer-doublet microtubules of the Chlamydomonas axoneme (Yang et al., 2000). We postulated that the axoneme must also contain a PP2A regulatory B-type subunit, responsible for localizing the heterotrimeric PP2A holoenzyme. To test this, we identified an axonemal PP2A B-subunit in the KCl fraction of the Chlamydomonas flagellar proteome (Pazour et al., 2005). The B-subunit gene predicts a protein that is homologous to the PP2A WD-repeat containing, PR55 family of B-subunits. Biochemical analyses confirmed that the B-subunit is an axonemal protein that it is extractable in 0.6 M NaCl. The B-subunit gene maps near the pf4 locus on LG I. The pf4 mutant cells have full-length flagella, display a smooth, medium velocity swimming phenotype and are defective in phototaxis. This phenotype is similar to mutant cells lacking inner arm I1 / f-dynein; however, I1 dynein is fully assembled in pf4 axonemes. Sequencing confirmed that pf4 is defective in the PP2A B-subunit gene: pf4 has a base pair deletion / substitution that results in a premature stop codon. Immunoblots revealed that the B-subunit protein is absent in pf4-mutant cells and axonemes. Additionally, the PP2A C-subunit is absent in pf4-mutant axonemes. Two independent, UV-induced pf4 revertants (pf4-V5, pf4-V11) were recovered from a genetic screen for suppressors and are tightly linked to pf4 (<0.27 mu and 0.24 mu, respectively). The pf4 revertants restore near wild-type swimming, normal phototaxis and assembly of PP2A B-subunit on to the axoneme. These results demonstrate that the axonemal B-subunit is required for targeting and anchoring PP2A in the axoneme and that PP2A is required for normal motility and phototaxis.

e-mail address of presenting author: win@cellbio.emory.edu

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