Fig. 1. Molecular characterization of Chlamydomonas FAP133. (A) Southern blot analysis of restricted Chlamydomonas genomic DNA, using the FAP133 cDNA as a probe. The blot reveals single bands in BamHI- and SmaI-digested samples indicating that there is a single FAP133 gene present in the Chlamydomonas genome. (B) Northern blot analysis of Chlamydomonas RNA demonstrating upregulation by 460% of the 2.4 kb FAP133 transcript 30 minutes after deflagellation (30'postDF) compared to non-deflagellated cells (NDF). The right panel shows the ethidium-bromide-stained gel used for the analysis; quantitation of the upper three bands revealed that the amount in the NDF sample was 78%, 99% and 72% that of the 30'postDF sample, respectively. (C) Neighbor-joining tree showing the relationship of Chlamydomonas FAP133 to other proteins containing WD-repeats. Phylogenetic analysis was based on a CLUSTALW alignment of FAP133 with Chlamydomonas outer-arm IC1 (Q39578) and IC2 (P27766), Chlamydomonas CrLis1 (ABG33844), mouse Lis1 (P63005), human G1 (P62873), rat cytoplasmic dynein DYNC1I2 (Q62871), human, Xenopus and Danio WD34 proteins (NM_052844, BC106359 and BC133909, respectively), and uncharacterized proteins from Strongylocentrotus purpuratus (XM_001197794), Leishmania infantum (XM_001466479), Trypanosoma brucei (XP_839051), Tetrahymena thermophila (XM_001022425), Paramecium tetraurelia (XM_001458772) and Tribolium castaneum (XM_966966). FAP133 is most closely related to the vertebrate WD34 proteins. (D) Sequence analysis of the Chlamydomonas FAP133 protein, using the SMART algorithm, revealed six WD-repeat domains that probably form a -propeller. Two degenerate putative LC8-binding sites, VETQT (residues 46-50) and QGTQT (residues 56-60), are located in the N-terminal part of the molecule.