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 1

Quantitative protein complex analysis of lebercilin.

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Quantitative protein complex analysis of lebercilin. (A) SILAC/AP approa...
(A) SILAC/AP approach to detect specific protein complex components. Identification of significantly enriched proteins compared with control (SF-TAP; green), and analysis of the effect of 2 LCA-causative mutations in lebercilin (p.P493TfsX1 and p.Q279X) on protein complex formation, by comparing the lebercilin complex with complexes formed by the mutants (red) in SILAC-labeled HEK293T cells. MS, mass spectrometry. (B) Detection of specific protein complex components. Plotted are log10 ratios and log10 intensities for each protein identified and quantified in at least 2 of 3 biological replicates. Significantly enriched proteins (P < 0.001) are plotted in green. (C) Protein complex of wild-type lebercilin. Gene names for each protein are shown (see Supplemental Table 1). Blue lines denote interconnectivity, as determined by IFT88 SF-TAP analysis (Supplemental Table 3). (D) Confirmation of IFT-lebercilin association by detecting endogenous lebercilin in eluates of IFT20, IFT27, IFT52, IFT57, and IFT88 SF-TAP experiments. Shown are the lysates as control for lebercilin input. All SF-TAP tagged constructs were expressed and purified, as demonstrated by Western blot using anti-FlagM2. Lebercilin was detected by anti-lebercilin (SN2135) in all eluates, but not in the SF-TAP control, confirming the SILAC/AP results. (E) Confirmation of the association of IFT and IFT-associated proteins with lebercilin in retina by detecting endogenous IFT88, IFT57, and TRAF3IP1 in a GST pulldown of lebercilin, but not in the GST control. Both GST-lebercilin and GST alone were expressed. Lanes for the anti-GST blot were run on the same gel but were noncontiguous (white line).