Mouse IMCD3 cells stably transfected with FLAG expression constructs were serum starved for 48 hr, fixed with paraformaldehyde and stained with DAPI (blue), FLAG (red) and IFT20 (green, left panel in each pair) or IFT27 (green, right panel in each pair). The transfected FLAG construct is listed on the left side of each set of panels. N and C refer the position of the tags. Cilia are marked with arrows. Insets are the FLAG (red) channels of the cilia to more clearly document the localization of the FLAG-tagged protein to this organelle. Note that all proteins except IFT172 (with either an N- or C-terminal tag), IFT20 and GFP localize to cilia. Also note that IFT20 is the only protein localized to the Golgi complex and that IFT54 displaces native IFT20 from the Golgi complex. Size bar is 10 μm and relevant for all panels.

Mouse IMCD3 cells transiently transfected with FLAG expression constructs were lysed 48 hrs after electroporation, immunoprecipitated with FLAG Ab and analyzed by western blotting. The FLAG-tagged bait protein is listed vertically at the top of the figure while name of the expression construct is listed vertically at the bottom. The left group is the starting material before immunoprecipitation and the right group is the precipitated material. The FLAG-tagged proteins are marked with arrows on the FLAG western. Note that GMAP210 is less abundant than the other proteins and is not visible at exposures reasonable for them. The right most lane (*) shows a longer exposure of immunoprecipitated GMAP210 from a different gel. Abs used for the western blots are listed on the right side. The western blot of IFT20 shows endogenous IFT20 at 15 kD and FLAG-IFT20 at 18 kD along with a degradation product of the FLAG-IFT20 that runs slightly faster. Note that the blank lane was inadvertently left out of the Rabaptin5 gel. To allow the lanes to line up, the image was split in the middle.

The finding that IFT20 is part of the Golgi IFT complex as well as complex B presents interesting possibilities for its function. This speculative model presents our view of how this may be occurring. IFT20 is anchored to the Golgi membrane by the golgin protein GMAP210 [Follit et al., 2008]. IFT20 is dynamic and moves from the Golgi complex to the cilium [Follit et al., 2006] but GMAP210 appears to remain at the Golgi. IFT20 binds directly to the complex B subunit IFT54 [this work and Omori et al. 2008] but IFT54 does not appear to localize to the Golgi complex and it is not part of the Golgi IFT complex [this work]. Thus, we hypothesize that IFT20 and GMAP210 function at the Golgi complex to sort proteins into vesicles destined for the ciliary membrane. These vesicles leave the Golgi with IFT20 associated with them. Once the vesicles arrive at the base of the cilium, IFT20 on the vesicles interacts with the IFT54 subunit of complex B to form the complete IFT complex. The vesicles fuse with the plasma membrane at the base of the cilium and the IFT complex with attached cargos is carried into the cilium by kinesin-2. By having the IFT complex assembled on the surface of the vesicle, the transport of membrane and non-membranous cargos could be coordinated.

Mouse IMCD3 cells stably transfected with FLAG expression constructs were lysed, immunoprecipitated with FLAG Ab and analyzed by western blotting. The FLAG-tagged bait protein is listed vertically at the top of the figure while name of the expression construct is listed vertically at the bottom. The left group is the starting material before immunoprecipitation and the right group is the precipitated material. The FLAG-tagged proteins are marked with arrows on the FLAG western. Abs used for the western blots are listed on the right side. The western blot of IFT20 shows endogenous IFT20 at 15 kD and FLAG-IFT20 at 18 kD along with a degradation product of the FLAG-IFT20 that runs slightly faster. Note that both IFT20 and C1orf41/IFT25 precipitated complex B subunits (IFT27, IFT88, IFT20) but not complex A (IFT140) or control proteins (PKD2).

Mouse IMCD3 cells stably transfected with FLAG expression constructs were lysed, immunoprecipitated with FLAG Ab and analyzed by western blotting. The FLAG-tagged bait protein is listed at the top of the figure. Abs used for the western blots are listed on the right side. Arrows indicate the FLAG-tagged protein. * at ~37 kD marks a non-specific protein that is immunoprecipitated by the FLAG antibody. Ladders are shown to the left of each of the gels. The sizes of the markers are the same for all and shown on the left most ladder. Input (left most lane of the first gel) is starting material from the IFT20 immunoprecipitation equivalent to 1/6 of the amount in the precipitated lane. Note that GFP-FLAG was much more abundant than the other proteins and 1/5 as much of this immunoprecipitation was loaded on the gel for the FLAG western. The amount was not altered in the gels for the IFT88, GMAP210 or IFT140 westerns.

A. Diagram summarizing data from experiments to map the IFT20 binding site on IFT54. Map illustrating the motif structure of IFT54 is shown on the top. Expression construct names are on the left. Exact amino acids are listed in the materials and methods. Arg rich domain is an unusual stretch of alternating positive and negatively charged residues. The coiled-coil domain is marked by series of circles. The IFT20 binding domain is marked by a solid dark bar. Summary of the activity of each of the constructs is shown on the right. B. IF of selected constructs to illustrate the ability of the C-terminal end of IFT54 (Red) to displace native IFT20 (green) from the Golgi complex. Left panel is a composite of red and green channels while the right panel shows only IFT20 staining in green. Arrows mark transfected cells. Arrowheads mark untransfected cells. Scale bar is 10 μm and relevant for all panels. C. Immunoprecipitation of selected constructs to illustrate the ability of C-terminal fragments of IFT54 to precipitate IFT20 but not IFT88. Mouse IMCD3 cells transiently transfected with FLAG expression constructs were lysed, immunoprecipitated with FLAG Ab and analyzed by western blotting. The FLAG-tagged expression construct is listed at the top of the figure. The left group is the starting material before immunoprecipitation and the right group is the precipitated material. The FLAG-tagged proteins are marked with arrows on the FLAG western. Antibodies used for western blotting are listed on the right. D. ClustalW alignment of the IFT20 binding site from IFT54 to a portion of the IFT20 binding site from GMAP210. * indicate identity, : and . mark similarity.