Molecular Basis of Tubulin Transport Within the Cilium by IFT74 and IFT81

Abstract

Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.Cilia are microtubule-based organelles that function in motility, sensory reception, and signaling (1). Ciliary dysfunction results in numerous diseases and disorders commonly known as ciliopathies. Intraflagellar transport (IFT) is involved in cilium formation (2, 3) but also functions in other cellular processes, such as the recycling of Tcell receptors at the immune synapse (4). IFT relies on kinesin-2 and IFT-dynein molecular motors moving along the microtubule-based axoneme of cilia (5-7) and on the IFT complex, which contains at least 20 different protein subunits. Although ~600 proteins are known to reside in the cilium (8), we know very little about how they are recognized as ciliary cargo by the IFT machinery (9-11).To identify potential cargo-binding sites on the IFT complex, we carried out bioinformatical and biochemical screening and identified conserved domains that were not required for IFT complex formation. We reasoned that such domains could protrude from the IFT particlecore structure and would thus be in a prime position for cargo recognition. The two IFT core (12). Given that the cilium consists of a MT-based axoneme, IFT of large quantities of tubulin is required for cilium formation (13). We thus tested the tubulin-binding properties of HsIFT81N using affinity pull-downs ( Fig. 1D and fig. S4E) and microscale thermophoresis (MST) with unpolymerized bovine αβ-tubulin (Fig. 1, E S5F). Thus, the tubulin-binding module is formed by the IFT74/81 complex rather than by IFT81N alone.To dissect the binding mode in the IFT74/81:αβ-tubulin complex, samples were prepared from MT and unpolymerized αβ-tubulin lacking the highly acidic C-terminal tails, often referred to as E-hooks (12) ( fig. S5A). αβ-tubulin lacking E-hooks had similar affinity for IFT81N as intact tubulin ( fig. S5, B and C), which suggested that IFT81N recognizes the globular domain of αβ-tubulin with no substantial interaction with the E-hooks. IFT74/81 displayed robust MT binding in sedimentation assays, which was, however, reduced to background levels in the absence of the β-tubulin E-hook ( fig. S5E). Thus, IFT81N appears to bind the globular domain of tubulin to provide specificity, and IFT74N recognizes the β-tubulin tail to increase affinity (Fig. 1H).To examine the role of tubulin binding by IFT74/81 in a cellular system, we transiently expressed Flag-HsIFT81 or Flag-HsIFT81ΔN in human RPE-1 cells and induced formation ...

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

“…The results of tubulin pull‐downs demonstrated that Mm IFT54CH binds tubulin regardless of the presence of E‐hooks, indicating that IFT54CH, like IFT81CH (Bhogaraju et al , 2013a), recognizes the globular domain of tubulin. It is currently not known whether IFT81CH and IFT54CH recognize the same or different binding sites on αβ‐tubulin.…”

“…Conserved residues are highlighted in yellow and residues that bind tubulin are marked blue for IFT 81 (Bhogaraju et al , 2013a) and green for IFT 54 (this study). The secondary structures for IFT 54 (magenta) and IFT 81 (green) are displayed above the sequences.…”

“…Mammalian IFT54 (known as TRAF3IP1 or MIPT3) and IFT81/74 were previously shown to associate with both soluble αβ‐tubulin and polymerized MTs (Ling & Goeddel, 2000; Bhogaraju et al , 2013a). We find that this is also the case for Chlamydomonas IFT54 as the IFT54/20 subcomplex co‐pelleted with MTs in a sedimentation assay (Appendix Fig S3E).…”

“…αβ‐tubulin assembles the backbone of the MT‐based axoneme of the cilium and is a known IFT cargo (Craft et al , 2015) that associates directly with IFT81/74 of the IFT‐B1 subcomplex (Bhogaraju et al , 2013a). The CH domain of IFT81 binds the globular part of αβ‐tubulin ( K d  = 16 μM), and mutations of tubulin‐binding residues significantly impair ciliogenesis in human cells (Bhogaraju et al , 2013a).…”

“…These include tubulin, the main axonemal building block, which is a cargo for IFT in both Chlamydomonas reinhardtii (Cr) (Craft et al , 2015) and C. elegans (Hao et al , 2011). In vitro studies suggested that tubulin cargo directly binds to the IFT complex via a dedicated module consisting of a calponin homology (CH) domain of IFT81 and a highly basic domain of IFT74 (Bhogaraju et al , 2013a). However, given that tubulin is the most abundant ciliary protein, it has been suggested that additional tubulin‐binding sites may exist within the IFT complex (Bhogaraju et al , 2014).…”