(A) Sequence alignment of deduced amino acid sequences from 13 invertebrate and vertebrate BBS3 orthologues and modified BBS3 sequences. Alignment was generated using CLC main workbench (version 6.8); the most conserved residues are shown in blue, the less conserved are in red. BBS3 contains five conserved domains including G1, G2, G3, G4, and G5, as labeled in the boxes. The P-loop threonine (T31) and alanine or glutamine (A73 or Q73) were marked. Dashes indicate gaps introduced to optimize the alignment. Arrowheads indicate missense mutations created in dominant-negative and constitutive-active BBS3 mutants. The BBS3 accession numbers are Bos Taurus, NP_001069250.1, Ovis aries, XP_004002902.1, Homo sapiens, AAH24239.1, Macaca mulatta, NP_001244689.1, Mus musculus, NP_062639.3, Rattus norvegicus, NP_001102312.1, Callorhinchus milii, XP_007899465.1, Xenopus tropicalis, XP_012813411.1, Danio rerio, NP_001008733.1, Drosophila melanogaster, NP_611421.1, Trypanosoma brucei, 228 .m00796, Trypanosoma cruzi, RNC37227.1, and Chlamydomonas reinhardtii, XP_001691012.1. (B) GTP hydrolysis of BBS3, BBS3A73L, and BBS3T31R. Introduction of a leucine at the position of A73 of BBS3 dramatically reduces the intrinsic GTPase activity, indicating that BBS3A73L is a GTP-locked, constitutive-active variant. No GTPase activity was observed for BBS3T31R, indicating that BBS3T31R appears to be an inactive GDP-locked or simply a dominant-negative variant.