Structure and activation mechanism of the BBSome membrane-protein trafficking complex

Abstract

Bardet-Biedl syndrome (BBS) is an incurable ciliopathy caused by the failure to correctly 1 establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS 2 assemble into the BBSome, a master regulator of the ciliary membrane proteome. We report the 3 electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and 4 active states at 3.1 and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to 5 an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive 7 state. Activation requires an unexpected swiveling of the b-propeller domain of BBS1, the key 8 subunit implicated in substrate recognition, which widens a central cavity of the BBSome. 9Structural mapping of disease-causing mutations suggests that pathogenesis predominantly 10 results from disruption of autoinhibition and activation. 11

The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells

“…However, in vitro experiments provided contradictory data concerning the sequence of the individual steps [18,21,22]. Although the resolution of the structure of the BBSome complex represented a break-through in the field [22][23][24], these studies could not reveal how the BBSome assembles in living cells, including the spatial regulation of individual steps. We generated a library of 64 RPE1-derived cell lines using a combination of CRISPR/Cas9 knockouts of individual BBSome subunits and retroviral expression of YFP-tagged subunits.…”

“…It is tempting to speculate that AZI1 and/or LZTFL1 mediate a quality control mechanism blocking the release of incomplete pre-BBSome from the PS. As BBS5 is a relatively peripheral subunit of the BBSome [22][23][24] that is sub-stoichiometrically incorporated into the complex in vitro [22], a control mechanism checking the incorporation of BBS5 into the pre-BBSome in cells seems to be plausible.…”

“…However, we cannot exclude that the last step of the BBSome formation is not the incorporation of BBS1 itself, but another BBS1-dependent event. It could be a conformational change in the BBSome, caused by a BBS1-interacting GTPase, BBS3/ARL6 [9,19,24]. In any case, BBS3 seems to be involved in the translocation of the (pre-)BBSome from the PS to the ciliary base, as the BBSome-BBS3-GTP does not co-purify with PCM-1 [3,9] and BBS3 was proposed to mediate the interaction of the BBSome with ciliary membranes [9,24].…”

“…It could be a conformational change in the BBSome, caused by a BBS1-interacting GTPase, BBS3/ARL6 [9,19,24]. In any case, BBS3 seems to be involved in the translocation of the (pre-)BBSome from the PS to the ciliary base, as the BBSome-BBS3-GTP does not co-purify with PCM-1 [3,9] and BBS3 was proposed to mediate the interaction of the BBSome with ciliary membranes [9,24]. BBS1 has been shown to interact with RABIN8, a guanine-exchange factor for RAB8, which is involved in vesicular trafficking and might regulate BBSome targeting to the ciliary base [3].…”

“…Three independent studies resolved the BBSome structure using cryo-EM with molecular modeling using the BBSome isolated from bovine retina [23] or using high-resolution cryo-EM analysis of bovine BBSome [24] or of human BBSome (lacking BBS2 and BBS7) expressed in insect cells [22]. These studies showed a high level of interconnectivity among the BBSome subunits, raising the question of how these BBSome proteins assemble in the cells.…”