Disruption of intraflagellar protein transport in photoreceptor cilia causes Leber congenital amaurosis in humans and mice
Karsten Boldt, … , Ronald Roepman, Marius Ueffing
Karsten Boldt, … , Ronald Roepman, Marius Ueffing
Published May 23, 2011
Citation Information: J Clin Invest. 2011;121(6):2169-2180. https://doi.org/10.1172/JCI45627.
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Research Article

Disruption of intraflagellar protein transport in photoreceptor cilia causes Leber congenital amaurosis in humans and mice

Abstract

The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.

Authors

Karsten Boldt, Dorus A. Mans, Jungyeon Won, Jeroen van Reeuwijk, Andreas Vogt, Norbert Kinkl, Stef J.F. Letteboer, Wanda L. Hicks, Ron E. Hurd, Jürgen K. Naggert, Yves Texier, Anneke I. den Hollander, Robert K. Koenekoop, Jean Bennett, Frans P.M. Cremers, Christian J. Gloeckner, Patsy M. Nishina, Ronald Roepman, Marius Ueffing

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Figure 2

Quantitative protein complex analysis of LCA-causative lebercilin mutations.

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Quantitative protein complex analysis of LCA-causative lebercilin mutati...
Detection of protein complex alterations by comparison of the complexes of SF-TAP tagged lebercilin to either lebercilin-p.P493TfsX1 (A) or lebercilin-p.Q279X (B) in HEK293T cells. Plotted are log10 ratios and log10 intensities as quantified by MaxQuant from 3 independent experiments. For each experiment, label switching was done to exclude label-specific effects. Shown are only proteins quantified and identified in at least 2 of 3 experiments. Highly significant (P < 0.001) altered protein interactions are plotted in red, the majority of them being IFT proteins (Supplemental Table 1). Also shown are representations of the respective protein complexes; all proteins of the IFT protein complex, as well as WDR26, KIAA0564, PGAM5, YPEL5, C20orf11, and RANBP9, were lost for both mutants. CSNK2A1/2, CSNK2B, DYNLL1, DYNLL2, and USP9X only lost association to lebercilin-p.Q279X, not to lebercilin-p.P493TfsX1.