Defects in Bardet-Biedl syndrome (BBS) genes impair Chlamydomonas phototaxis
Karl-Ferdinand Lechtreck and George B. Witman
Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655
BBS is a ciliopathy characterized by blindness, kidney abnormalities, and obesity. It is caused by defects in the BBSome, which contains BBS1, 2, 4, 5, 7, 8 and 9, is highly conserved, and is present in the flagella of Chlamydomonas. The non-phototactic Chlamydomonas mutants ptx5 and ptx6 have defects in BBS4 and BBS7, respectively. Screening of 16 non-phototactic mutants with good motility identified a bbs1 mutant and six uncharacterized mutants that lack BBS4 protein in their flagella. Therefore, the Chlamydomonas BBSome has a function that is important for phototaxis. Simultaneous total internal reflection fluorescence microscopy of BBS4-GFP and the intraflagellar transport (IFT) particle protein IFT20 tagged with mCherry revealed that the BBSome undergoes IFT in association with a subset of IFT particles. Signaling proteins including two protein kinases (AMPK and CAMKK) and phospholipase D (PLD) are abnormally enriched in bbs flagella, and the levels of several of these proteins increase over time in bbs but not wild-type flagella. These results suggest that the BBSome functions as an IFT cargo adapter for the export of signaling proteins from the flagellum. Phototactic behavior was transiently improved in bbs4 cells with newly regenerated flagella but reverted to the non-phototactic phenotype as the flagella aged, suggesting that the accumulation of the signaling proteins is disrupting phototaxis. AMPK, CAMKK, and PLD lack transmembrane domains but are predicted to be palmitoylated and myristoylated, indicative of a reversible association with ordered membrane domains. Metabolic labeling with myristic acid revealed that the bbs4 flagellar membrane has increased amounts of myristoylated proteins. Therefore, the BBSome is required to maintain and adjust the amounts of membrane (possibly lipid-raft-)-associated signaling proteins in the flagellum. We propose that BBSome loss affects flagellar signaling protein homeostasis, impairing phototaxis in Chlamydomonas and causing BBS in humans.
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