Assay for in vitro budding of ciliary-targeted rhodopsin transport carriers

Primary cilia and cilia-derived sensory organelles are cell's antennas that contain sensory receptors and signal transduction modules. Defects in the expression and targeting of ciliary proteins to this specialized cellular compartment lead to human disorders collectively known as ciliopathies. To examine the molecular basis for the ciliary targeting of the light receptor rhodopsin, we have developed a cell-free assay that reconstitutes its packaging into the specific post-Golgi rhodopsin transport carriers (RTCs). This assay accurately reproduces the in vivo process of carrier budding, while allowing examination of individual components of the macromolecular complexes, thus providing insight into a more general mechanism for the regulation of ciliary membrane targeting. Examples are shown for the use of this assay in rhodopsin trafficking. The cell-free assay is applicable to other ciliary-targeted sensory molecules.