The Tre2-Bub2-Cdc16 (TBC) domain-containing RAB-specific GTPase-activating proteins (TBC/RABGAPs) are characterized by the presence of highly conserved TBC domains and act as negative regulators of RABs. The importance of TBC/RABGAPs in the regulation of specific intracellular trafficking routes is now emerging, as is their role in different diseases. Importantly, TBC/RABGAPs act as key regulatory nodes, integrating signalling between RABs and other small GTPases and ensuring the appropriate retrieval, transport and delivery of different intracellular vesicles.

Since the publication of this article, the authors have further optimized the sequence analysis of the RABGAP TBC domains in Supplementary information S1 (figure), using more stringent criteria that also include the homology of the sequences surrounding the catalytic fingers. This has allowed them to identify a consensus sequence motif and to redefine the proposed grouping of TBC/RABGAPs in BOX 1 and Supplementary information S2 (table). A revised version of BOX 1 is shown below. Box 1 | Classical and unconventional TBC/RABGAPsThe presence of a dual-finger mechanism is, to our current knowledge, a unique feature of the Tre2-Bub2-Cdc16 (TBC) domain, which uses two catalytic residues, R and Q, to stabilize the transition state and catalyse the GTP hydrolysis of RABs 72. Analysis of TBC domain-containing RAB-specific GTPase-activating proteins (TBC/ RABGAPs) revealed that they predominantly share six conserved regions (A-F). A consensus sequence motif is deduced from the conserved regions B and C (Supplementary information S1 (figure)): it emphasizes two distinct signatures containing R and Q fingers found in 29 family members. These proteins are known as classical TBC/ RABGAPs (group 1: RQ; see table). Interestingly, 13 unconventional TBC/RABGAPs lacking the R and/or Q fingers have been identified (see below). If these unconventional TBC/RABGAPs do inactivate RABs, they probably use a different mode of action.We propose to group these unconventional TBC/RABGAPs into four groups according to the deviations of the catalytic fingers (see the table). The first three proposed groups comprise: TBC1 domain family members 29 and 13 (TBC1D29 and TBC1D13; group 2: RX), transferrin receptor-like 2 (TRE2; group 3: RR), TBC1D3 and TBC1D26 (group 4: XR). These proteins share conserved regions within the TBC domains with the classical TBC/RABGAPs (see Supplementary information S1 ( figure)). By contrast, the members of the last proposed group of TBC/RABGAPs lack the RQ finger (group 5: XX) and show poor sequence similarity to the classical TBC domains, especially within the conserved regions B and C. Furthermore, a few proteins have incomplete TBC domains .With two exceptions, no substrate has yet been reported for these unconventional TBC/RABGAPs (see Supplementary information S2 (table)). TBC1D3 (group 4: XR) undergoes a persistent interaction with RAB5 and is thus thought to be an effector of this RAB 51 . Taken together, the homology within the TBC regions (A-F) and the fact that RQ fingers are indispensable catalytic residues 36 suggest that the proteins in groups RX, RR, XR and XX may be inactive TBC/RABGAPs. Interestingly, out of the eight proteins in group XX, TBC1D7 has been reported to be a GAP for RAB17 (REF. 8), indicating that this group might work through an alternative mechanism to stimulate GTP hydrolysis. Nevertheless, an inactive TBC domain may still have important cellular functions to coordinate different signalling pathways, as is observed with TBC1D3 and TRE2 (see main text) and, similarly, with the RHOGAP-...

The BAR (Bin/amphiphysin/Rvs) domain defines an emerging superfamily of proteins implicated in fundamental biological processes by sensing and inducing membrane curvature. We identified a novel autoregulatory function for the BAR domain of two related GAPs' (GTPase-activating proteins) of the GRAF (GTPase regulator associated with focal adhesion kinase) subfamily. We demonstrate that the N-terminal fragment of these GAPs including the BAR domain interacts directly with the GAP domain and inhibits its activity. Analysis of various BAR and GAP domains revealed that the BAR domain-mediated inhibition of these GAPs' function is highly specific. These GAPs, in their autoinhibited state, are able to bind and tubulate liposomes in vitro, and to generate lipid tubules in cells. Taken together, we identified BAR domains as cis-acting inhibitory elements that very likely mask the active sites of the GAP domains and thus prevent down-regulation of Rho proteins. Most remarkably, these BAR proteins represent a dual-site system with separate membrane-tubulation and GAP-inhibitory functions that operate simultaneously.

RHO GTPase-activating proteins (RHOGAPs) are one of the major classes of regulators of the RHO-related protein family that are crucial in many cellular processes, motility, contractility, growth, differentiation, and development. Using database searches, we extracted 66 distinct human RHOGAPs, from which 57 have a common catalytic domain capable of terminating RHO protein signaling by stimulating the slow intrinsic GTP hydrolysis (GTPase) reaction. The specificity of the majority of the members of RHOGAP family is largely uncharacterized. Here, we comprehensively investigated the sequence-structurefunction relationship between RHOGAPs and RHO proteins by combining our in vitro data with in silico data. The activity of 14 representatives of the RHOGAP family toward 12 RHO family proteins was determined in real time. We identified and structurally verified hot spots in the interface between RHOGAPs and RHO proteins as critical determinants for binding and catalysis. We have found that the RHOGAP domain itself is nonselective and in some cases rather inefficient under cell-free conditions. Thus, we propose that other domains of RHOGAPs confer substrate specificity and fine-tune their catalytic efficiency in cells.