iTRAQ analysis of lf2 mutant flagella identifies new candidate proteins involved in IFT and flagellar length control
Lai-Wa Tam and Paul A. Lefebvre
Plant Biology, University of Minnesota, 1445 Gortner Ave., St Paul, MN 55108, USA
The length of flagella in Chlamydomonas is tightly regulated by signal transduction mechanisms involving several different kinases. One of these kinases is encoded by the Long Flagella 2 (LF2) gene. To characterize biochemical changes associated with loss of flagellar length control, we used a new technique called iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) to compare the protein composition of wild-type flagella with flagella of two different lf2 mutants. Different lf2 mutant alleles exhibit varying degrees of defective flagellar length control and flagellar assembly. lf2-5 is a hypomorphic allele with abnormally long flagella, whereas lf2-1 is a null allele that often shows two flagella of different lengths on a single cell. More importantly, many lf2-1 flagella have swellings at their tips that are filled with piled-up strings of what appear to be intraflagellar transport (IFT) protein particles. As expected from our observation that lf2-1 flagella over-accumulate IFT particles, iTRAQ analysis identified many of the known IFT proteins from both complex A and complex B that were enriched only in the lf2-1 mutant flagella. A number of uncharacterized flagellar proteins also displayed similar enrichment profile in lf2-1 flagella, indicating that this approach has the potential to discover new candidate proteins for IFT that have not been previously identified. In addition, we also identified a number of interesting flagellar proteins that are enriched or depleted in both lf2 mutants. These proteins are prime candidates as targets for the regulation of flagellar length and assembly by LF2.
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