Summary The BBSome is a complex of Bardet-Biedl Syndrome (BBS) proteins that shares common structural elements with COPI, COPII and clathrin coats. Here we show that the BBSome constitutes a coat complex that sorts membrane proteins to primary cilia. Biochemically, the BBSome is the major effector of the Arf-like GTPase Arl6/ BBS3. In vivo, the BBSome and Arl6 localize to ciliary punctae and Arl6GTP is required to target the BBSome to cilia. Congruently, GTP-bound Arl6 and acidic phospholipids are sufficient to efficiently recruit the BBSome to chemically defined liposomes. Finally, ultrastructural analyses demonstrate that BBSome binding to liposomes produces distinct patches of polymerized coat. Since we establish that the ciliary targeting signal of somatostatin receptor 3 needs to be directly recognized by the BBSome to mediate targeting to cilia, we propose that trafficking to cilia entails the coupling of BBSome coat polymerization to the recognition of sorting signals.

Spastin, an AAA ATPase mutated in the neurodegenerative disease hereditary spastic paraplegia, severs microtubules. Many other AAA proteins form ring-shaped hexamers and contain pore loops, which project into the ring's central cavity and act as ratchets that pull on target proteins, leading, in some cases, to conformational changes. We show that Spastin assembles into a hexamer and that loops within the central pore recognize C-terminal amino acids of tubulin. Key pore loop amino acids are required for severing, including one altered by a disease-associated mutation. We also show that Spastin contains a second microtubule binding domain that makes a distinct interaction with microtubules and is required for severing. Given that Spastin engages the MT in two places and that both interactions are required for severing, we propose that severing occurs by forces exerted on the C-terminal tail of tubulin, which results in a conformational change in tubulin, which releases it from the polymer.

AAA1 adenosine triphosphatases (ATPases) are molecular machines that perform a wide variety of cellular functions. For instance, they can act in vesicle transport, organelle assembly, membrane dynamics and protein unfolding. In most cases, the ATPase domains of these proteins assemble into active ring-shaped hexamers. As AAA1 proteins have a common structure, a central issue is determining how they use conserved mechanistic principles to accomplish specific biological actions. Here, we review the features and motifs that partially define AAA1 domains, describe the cellular activities mediated by selected AAA1 proteins and discuss the recent work, suggesting that various AAA1 machines with very different activities employ a common core mechanism. The importance of this mechanism to human health is demonstrated by the number of genetic diseases caused by mutant AAA1 proteins.

Highlights d 4.9-Å density map of the intact native BBSome obtained by single-particle cryo-EM d Ca model generated using Rosetta establishes the subunit organization d BBSome subunits have a very high degree of interconnectivity d In solution, the BBSome exists predominantly in an autoinhibited state

Hereditary spastic paraplegia (HSP) is a motor neuron disease caused by a progressive degeneration of the motor axons of the corticospinal tract. Point mutations or exon deletions in the microtubule-severing ATPase, spastin, are responsible for approximately 40% of cases of autosomal dominant HSP. Here, we report the 3.3 Å X-ray crystal structure of a hydrolysis- deficient mutant (E442Q) of the human spastin protein AAA domain. This structure is analyzed in the context of the existing Drosophila melanogaster spastin AAA domain structure and crystal structures of other closely related proteins in order to build a more unifying framework for understanding the structural features of this group of microtubule-severing ATPases.