Abstract
The cilium is an organelle used for motility and cellular signaling. Intraflagellar transport (IFT) is a process to move ciliary building blocks and signaling components into the cilium. How IFT controls the movement of ciliary components is currently poorly understood. IFT172 is the largest IFT subunit essential for ciliogenesis. Due to its large size, the characterization of IFT172 has been challenging. Using giant unilamellar vesicles (GUVs), we show that IFT172 is a membrane-interacting protein with the ability to remodel large membranes into small vesicles. Purified IFT172 has an architecture of two globular domains with a long rod-like protrusion, resembling the domain organization of coatomer proteins such as COPI-II or clathrin. IFT172 adopts two different conformations that can be manipulated by lipids or detergents: 1) an extended elongated conformation and 2) a globular closed architecture. Interestingly, the association of IFT172 with membranes is mutually exclusive with IFT57, implicating multiple functions for IFT172 within IFT.
Highlights
The cilium is an organelle used for motility and cellular signaling
IFT172 expressed with a C-terminal His-tag appeared in the soluble fraction after centrifugation of the cell lysate, but the elution from Ni-NTA beads resulted in a turbid white solution indicative of a high lipid content (Supplementary Table1)
While we have previously used purified IFT17223 that eluted at an estimated molecular weight of around 400 kDa in size exclusion chromatography (SEC), a significant fraction of IFT172 eluted in the void volume suggesting a molecular weight of more than 1 M Da (Fig. 1b)
Summary
The cilium is an organelle used for motility and cellular signaling. Intraflagellar transport (IFT) is a process to move ciliary building blocks and signaling components into the cilium. IFT proteins were observed on vesicles destined for the cilium in Chlamydomonas[18] and membrane shedding from cilia was observed for a number of organisms and cell types including Chlamydomonas and C.elegans[19,20,21]. These observations suggest that subunits of the IFT complex subunits may have affinity for membranes the molecular mechanisms of such IFTmembrane interactions remain elusive. Larger foci formation of IFT172 can be observed at the initiation stage of ciliogenesis, implying the IFT172-lipids association has a role during the cilia formation
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