Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

Abstract

Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT‐B complex consists of 9–10 stably associated core subunits and six “peripheral” subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six “peripheral” IFT‐B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT‐B core. We suggest a nomenclature of IFT‐B1 (core) and IFT‐B2 (peripheral) for the two IFT‐B subcomplexes. We demonstrate that IFT88, together with the N‐terminal domain of IFT52, is necessary to bridge the interaction between IFT‐B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT‐B1/IFT‐B2 complex formation. Furthermore, we show that of the three IFT‐B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ‐tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface‐exposed residues.

IFT57 stabilizes the assembled intraflagellar transport complex and mediates transport of motility-related flagellar cargo

“…Research in the past decade has made great advances into determining the architecture of the IFT complexes, especially the IFT-B complex Taschner et al, 2016;Katoh et al, 2016). The IFT-B complex contains a salt-resistant core complex and several peripheral proteins (Lucker et al, 2005).…”

“…Indeed, two very recent comprehensive mappings of interactions among all IFT-B subunits demonstrate that all peripheral proteins [IFT20, IFT54 (also known as TRAF3IP1), DYF-3 (also known as Cluap1), IFT57, IFT80 and IFT172] form a distinct biochemical complex which is now called IFT-B2. The core complex, containing IFT22, IFT25 (also known as HSPB11), IFT27, IFT46, IFT52 (also known as BLD1), IFT56 (also known as TTC26 and DYF-13), IFT70 (also known as DYF-1), IFT74, IFT81 and IFT88, is renamed as IFT-B1 (Katoh et al, 2016;Taschner et al, 2016).…”

“…IFT52 is also essential for the overall stability of IFT-B1 because it mediates the binding of the subcomplex IFT88-IFT70-IFT52-IFT46 with the other subcomplex IFT81-IFT74-IFT27-IFT25-IFT22 (Taschner et al, 2014(Taschner et al, , 2011. Recently, the Lorentzen and the Nakayama laboratories, using expressed Chlamydomonas reinhardtii, and human or mouse proteins, respectively, have biochemically mapped the subunits at the interface between IFT-B1 and IFT-B2 (Taschner et al, 2016;Katoh et al, 2016). IFT57 and DYF-3 in IFT-B2, and IFT52 and IFT88 in IFT-B1 are the proteins that bridge the two subcomplexes together to form a complete IFT-B complex.…”

Structural and Functional Recovery of Sensory Cilia in C. elegans IFT Mutants upon Aging

“…However, it is unlikely that simple genetic buffering via upregulation of HSF1/Hsp90 during aging is sufficient for AdCR since neither overexpression of HSF1 nor induction of the heat shock response in 1d old osm-6 mutants is sufficient to suppress their ciliary defects. Instead, we speculate that in younger animals, expression of a partly functional IFT protein in the absence of the wildtype protein disrupts IFT complex function [35,36,79,80]. Reduced levels of mutant protein in aged animals via increased UPS activity, coupled with chaperone-mediated stabilization of the complex or folding intermediates enables productive IFT and AdCR in IFT hypomorphic mutant animals ( S6 Fig). HSF-1/Hsp90 and UPS may also indirectly affect IFT to improve ciliogenesis.…”

“…Compromised proteostasis in aged animals is in part due to reduced functionality of protein quality control mechanisms, thereby enhancing aggregation and accumulation of misfolded proteins [30][31][32][33][34]. Thus, protein complexes such as IFT particles that rely on defined stoichiometry of individual components [3,35,36] may be particularly vulnerable to aging. However, how aging affects primary cilia structure and function has not been examined in detail.…”