Primary cilia are generated through the extension of the microtubule-based axoneme. Centrosomal protein 104 (CEP104) localizes to the tip of the elongating axoneme, and CEP104 mutations are linked to a ciliopathy, Joubert syndrome. Thus, CEP104 has been implicated in ciliogenesis. However, the mechanism by which CEP104 regulates ciliogenesis remains elusive. We report here that CEP104 is critical for cilium elongation but not for initiating ciliogenesis. We also demonstrated that the tumor-overexpressed gene (TOG) domain of CEP104 exhibits microtubule-polymerizing activity and that this activity is essential for the cilium-elongating activity of CEP104. Knockdown/rescue experiments showed that the N-terminal jelly-roll (JR) fold partially contributes to cilium-elongating activity of CEP104, but neither the zinc-finger region nor the SXIP motif is required for this activity. CEP104 binds to a centriole-capping protein, CP110, through the zinc-finger region and to a microtubule plus-end-binding protein, EB1, through the SXIP motif, indicating that the binding of CP110 and EB1 is dispensable for the cilium-elongating activity of CEP104. Moreover, CEP104 depletion does not affect CP110 removal from the mother centriole, which suggests that CEP104 functions after the removal of CP110. Last, we also showed that CEP104 is required for the ciliary entry of Smoothened and export of GPR161 upon Hedgehog signal activation and that the TOG domain plays a critical role in this activity. Our results define the roles of the individual domains of CEP104 in its functions in cilium elongation and Hedgehog signaling and should enhance our understanding of the mechanism underlying CEP104 mutation-associated ciliopathies.BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and kinetochores, much less is known regarding BuGZ’s requirements. Here, we used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, we found that the kinetics of Bub1, BubR1, and BuGZ loading to kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to kinetochores, we carried out size-exclusion chromatography and analyzed Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions. We found that prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. Our results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling.Powered by the energy of ATP binding and hydrolysis, protease-containing ABC transporters (PCATs) export amphipathic and hydrophilic bacteriocin and quorum-sensing proteins across the membrane hydrophobic barrier. The cargo proteins have N-terminal leader peptides that are cleaved off by the cysteine protease domain, referred to as the C39 domain, or referred to as the peptidase (PEP) domain. The sequence and structural determinants of the interaction between PCATs and cargo proteins are poorly understood, yet this interaction is a central aspect of the transport mechanism. Here, we demonstrate the ATP-dependent, equilibrium binding of the cargo protein to the transmembrane domain (TMD) of a PCAT subsequent to the removal of the leader peptide by the PEP domain. Binding of the cargo protein to PCAT1 variants devoid of the PEP domain is detected through changes in the spectroscopic properties of fluorescent or spin label. see more Moreover, we find similar energetics of binding regardless of the presence of the leader peptide, suggesting that although the PEP domain serves for recognition and orientation, interaction with the TMD is the main contributor to the affinity. These findings are in direct contradiction with a recent study claiming that the TMD does not interact with the cargo protein; rather acting as a “Teflon-like” conduit across the bilayer (Kieuvongngam, V., Olinares, P. D. B., Palillo, A., Oldham, M. L., Chait, B. T., and Chen, J. (2020) Structural basis of substrate recognition by a polypeptide processing and secretion transporter. eLife 9, e51492). A distinctive feature of the transport model emerging from our data invokes a stable complex between PCATs and their cargo proteins following processing of the leader peptide and prior to ATP-dependent alternating access that translocates the cargo protein to the extracellular side.Excitatory amino acid transporters (EAATs) are prototypical dual function proteins that function as coupled glutamate/Na+/H+/K+ transporters and as anion-selective channels. Both transport functions are intimately intertwined at the structural level Secondary active glutamate transport is based on elevator-like movements of the mobile transport domain across the membrane, and the lateral movement of this domain results in anion channel opening. This particular anion channel gating mechanism predicts the existence of mutant transporters with changed anion channel properties, but without alteration in glutamate transport. We here report that the L46P mutation in the human EAAT2 transporter fulfills this prediction. L46 is a pore-forming residue of the EAAT2 anion channels at the cytoplasmic entrance into the ion conduction pathway. In whole-cell patch clamp recordings, we observed larger macroscopic anion current amplitudes for L46P than for WT EAAT2. Rapid l-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters.