Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17

Abstract

Intraflagellar transport (IFT) is a microtubule based system that supports the assembly and maintenance of cilia. Genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the IFT machinery. In this study we prepared mouse pituitary cells that expressed an epitope-tagged IFT protein and immuno-purified the IFT B complex from these cells. Mass spectrometry analysis of the isolated complex led to identification of a number of well known components of the IFT B complex. In addition, peptides corresponding to mouse tetratricopeptide repeat proteins, TTC30A1, TTC30A2 and TTC30B were identified. The mouse Ttc30A1, Ttc30A2, Ttc30B genes are orthologs of Caenorhabditis elegans dyf-1, which is required for assembly of the distal segment of the cilia. We used co-immunoprecipitation studies to provide evidence that, TTC30A1, TTC30A2 or TTC30B can be incorporated into a complex with a known IFT B protein, IFT52. We also found that TTC30B can interact with mouse KIF17, a kinesin which participates in IFT. In vitro expression in a cell-free system followed by co-immunoprecipitation also provided evidence that TTC30B can directly interact with several different IFT B complex proteins. The findings support the view that mouse TTC30A1, TTC30A2 and TTC30B can contribute to the IFT B complex, likely through interactions with multiple IFT proteins and also suggest a possible link to the molecular motor, KIF17 to support transport of cargo during IFT.

The Intraflagellar Transport Machinery

“…Interaction studies of IFT-B core proteins using yeast two-hybrid analysis or bacterial coexpressions/pulldowns suggested direct interactions between IFT27 and IFT25 (Follit et al 2009;Wang et al 2009;Bhogaraju et al 2011), IFT70 andIFT52 (Zhao andMalicki 2011;Howard et al 2013), IFT70 and IFT46 (Fan et al 2010), IFT81 and IFT74 (Lucker et al 2005;Kobayashi et al 2007), IFT88, IFT52, and IFT46 (Lucker et al 2010), and, finally, IFT56 and IFT46 (Swiderski et al 2014). These initial insights into subunit interactions paved the way for the recombinant reconstitution of several IFT-B core subcomplexes using Chlamydomonas proteins ) as well as a nine-subunit IFT-B core lacking only IFT56 (Taschner et al 2014).…”

“…Regarding direct attachment points between motors and cargos, it was found that Dyf-1 in C. elegans (the worm homolog of the IFT-B1 protein IFT70) is necessary for binding and/or activation of the OSM-3 motor, because in dyf-1 mutants the IFT trains are moved only by heterotrimeric kinesin II, and the OSM-3 dependent distal singlet segment fails to form (Ou et al 2005a). A direct interaction between the homologous proteins in mammals was suggested (Howard et al 2013).…”

Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery

“…In C. elegans amphid channel cilia, heterotrimeric kinesin‐2 (aka kinesin‐II) appears to bind and transport IFT‐A, whereas homodimeric kinesin‐2 (OSM‐3) binds IFT‐B, because in BBSome mutants, the kinesin‐II/IFT‐A and OSM‐3/IFT‐B subcomplexes move separately along the axoneme at 0.5 and 1.2 μm·s −1 respectively . The IFT‐B1 subunit, IFT‐70 (DYF‐1) was proposed to be involved in the IFT‐B/OSM‐3 interaction, which is supported by coimmunoprecipitation experiments in mouse, suggesting that IFT70 binds to multiple IFT‐B subunits and to homodimeric kinesin‐2 (KIF17) . In contrast to the findings from C. elegans however, coimmunoprecipitation experiments in Chlamydomonas (which lacks homodimeric kinesin‐2) and mouse suggest that heterotrimeric kinesin‐2 binds directly to IFT‐B rather than IFT‐A , but the significance of this difference is unclear.…”

Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

“…The inability of KIF17 to rescue ciliogenesis was surprising as mammalian KIF17 is an active kinesin motor [73] that localizes to mammalian cilia [74], binds to IFT proteins [46, 75], moves in an IFT-like manner in mammalian cilia [45, 76], and influences the ciliary localization of some signaling proteins [39, 77]. We thus considered the possibility that heterotrimeric KIF3A/KIF3B/KAP plays a critical role during cilium assembly, but that KIF17 is able to drive sufficient IFT events for cilium maintenance.…”