Characterization of the Intraflagellar Transport Complex B Core

Abstract

Required for the assembly and maintenance of eukaryotic cilia and flagella, intraflagellar transport (IFT) consists of the bidirectional movement of large protein particles between the base and the distal tip of the organelle. Anterograde movement of particles away from the cell body is mediated by kinesin-2, whereas retrograde movement away from the flagellar tip is powered by cytoplasmic dynein 1b/2. IFT particles contain multiple copies of two distinct protein complexes, A and B, which contain at least 6 and 11 protein subunits, respectively. In this study, we have used increased ionic strength to remove four peripheral subunits from the IFT complex B of Chlamydomonas reinhardtii, revealing a 500-kDa core that contains IFT88, IFT81, IFT74/72, IFT52, IFT46, and IFT27. This result demonstrates that the complex B subunits, IFT172, IFT80, IFT57, and IFT20 are not required for the core subunits to stay associated. Chemical cross-linking of the complex B core resulted in multiple IFT81-74/72 products. Yeast-based two-hybrid and three-hybrid analyses were then used to show that IFT81 and IFT74/72 directly interact to form a higher order oligomer consistent with a tetrameric complex. Similar analysis of the vertebrate IFT81 and IFT74/72 homologues revealed that this interaction has been evolutionarily conserved. We hypothesize that these proteins form a tetrameric complex, (IFT81) 2 (IFT74/72) 2 , which serves as a scaffold for the formation of the intact IFT complex B.

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

“…LC‐MS/MS analysis of eluates demonstrated that IFT172 but not other IFT subunits are significantly enriched vs. the negative control suggesting that IFT57 1–234 ‐GST specifically captures IFT172 via a salt‐dependent interaction (Appendix Fig S4). The finding that IFT172 is only loosely attached to the IFT complex is in agreement with previously published results from sucrose gradient centrifugations of natively purified Chlamydomonas IFT‐B complexes (Lucker et al , 2005). On the other hand, our observation that IFT88/52 remains associated with an IFT‐B2 complex consisting of IFT80/57/54/38/20 at even 1 M NaCl is somewhat surprising as the “peripheral” IFT‐B2 subunits were previously reported to dissociate at 300 mM NaCl concentration from the IFT‐B1 complex (Lucker et al , 2005).…”

“…IFT172 and IFT80 are both predicted to contain two N‐terminal WD40 β‐propeller domains followed by α‐solenoid structure akin to the domain architecture observed for coatomer subunits (van Dam et al , 2013). Mutations in IFT172 and IFT80 were reported to result in skeletal pattering defects (Beales et al , 2007; Halbritter et al , 2013), and the two components interact genetically (Halbritter et al , 2013) albeit not physically in sucrose gradients (Lucker et al , 2005). We observed no direct interaction between IFT172 and IFT80 in SEC (Appendix Fig S2A and B).…”

“…The IFT complex organizes into a 6 subunit IFT‐A and a 16 subunit IFT‐B subcomplex that separate in sucrose gradients even at low salt concentrations (Piperno & Mead, 1997; Cole et al , 1998; Lucker et al , 2005; Omori et al , 2008; Follit et al , 2009; Lechtreck et al , 2009b; Fan et al , 2010; Taschner et al , 2011; Ishikawa et al , 2014). Nine of the IFT‐B subunits (IFT88/81/74/70/52/46/27/25/22) were shown to assemble into a stable IFT‐B core (Lucker et al , 2005; Taschner et al , 2014).…”

“…Nine of the IFT‐B subunits (IFT88/81/74/70/52/46/27/25/22) were shown to assemble into a stable IFT‐B core (Lucker et al , 2005; Taschner et al , 2014). IFT56 was recently characterized as an IFT‐B complex protein involved in the transport of axonemal motility factors (Ishikawa et al , 2014), and yeast‐2‐hybrid and pull‐down analyses suggest that it may associate with the IFT‐B core complex (Swiderski et al , 2014).…”

“…IFT56 was recently characterized as an IFT‐B complex protein involved in the transport of axonemal motility factors (Ishikawa et al , 2014), and yeast‐2‐hybrid and pull‐down analyses suggest that it may associate with the IFT‐B core complex (Swiderski et al , 2014). The six remaining IFT‐B proteins (IFT172, IFT80, IFT57, IFT54, IFT38, and IFT20) are referred to as “peripheral” subunits as they were shown to dissociate from the core at 300 mM NaCl concentration (Lucker et al , 2005). Despite the label as peripheral, several lines of evidence in the literature suggest that these IFT‐B subunits are as important for IFT and ciliogenesis as the IFT‐B core factors.…”