ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS. ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics.

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 ...

Eukaryotic cilia and flagella are evolutionarily conserved organelles that protrude from the cell surface. The unique location and properties of cilia allow them to function in vital processes such as motility and signaling. Ciliary assembly and maintenance rely on intraflagellar transport (IFT), the bidirectional movement of a multicomponent transport system between the ciliary base and tip. Since its initial discovery more than two decades ago, considerable effort has been invested in dissecting the molecular mechanisms of IFT in a variety of model organisms. Importantly, IFT was shown to be essential for mammalian development, and defects in this process cause a number of human pathologies known as ciliopathies. Here, we review current knowledge of IFTwith a particular emphasis on the IFT machinery and specific mechanisms of ciliary cargo recognition and transport.

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.

Crystallographic analysis of the B core complex of the intraflagellar transport machinery provides insight into the molecular basis of ciliogenesis defects caused by several specific IFT mutations.