The Molecular Architecture of Native BBSome Obtained by an Integrated Structural Approach.

The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-Å resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data. We find that BBSome subunits have a very high degree of interconnectivity, explaining the obligate nature of the complex. Furthermore, like other coat adaptors, the BBSome exists in an autoinhibited state in solution and must thus undergo a conformational change upon recruitment to membranes by the small GTPase ARL6/BBS3. Our model provides the first detailed view of the machinery enabling ciliary exit.

Crystal structure of the human spastin AAA domain.

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.

The conserved Bardet-Biedl syndrome proteins assemble a coat that traffics membrane proteins to cilia.

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. The BBSome is the major effector of the Arf-like GTPase Arl6/BBS3, and the BBSome and GTP-bound Arl6 colocalize at ciliary punctae in an interdependent manner. Strikingly, Arl6(GTP)-mediated recruitment of the BBSome to synthetic liposomes produces distinct patches of polymerized coat apposed onto the lipid bilayer. Finally, the ciliary targeting signal of somatostatin receptor 3 needs to be directly recognized by the BBSome in order to mediate targeting of membrane proteins to cilia. Thus, we propose that trafficking of BBSome cargoes to cilia entails the coupling of BBSome coat polymerization to the recognition of sorting signals by the BBSome.

AAA+ ATPases: achieving diversity of function with conserved machinery.

AAA+ 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 AAA+ 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 AAA+ domains, describe the cellular activities mediated by selected AAA+ proteins and discuss the recent work, suggesting that various AAA+ 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 AAA+ proteins.

Recognition of C-terminal amino acids in tubulin by pore loops in Spastin is important for microtubule severing.

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.