Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes
Fig 1
TBC-2 interacts physically with RAB-10 and AMPH-1.
(A) and (B) The interaction between TBC-2 and RAB-10(Q68L) requires a segment of TBC-2(AA 279–321) containing a predicted coiled-coil domain. RAB-10(Q68L) was expressed in a yeast reporter strain as a fusion to the DNA-binding domain of LexA (bait). Different truncations of TBC-2 were expressed in the same yeast cells as fusions to the B42 transcriptional activation domain (prey). Interaction between bait and prey was assayed by complementation of leucine auxotrophy (LEU2 growth assay). Colonies were diluted in liquid and spotted on solid growth medium directly (1X) or after further dilution (0.1X). (C) Alanine substitutions within the critical RAB-10-binding sequence of TBC-2 (positions aa283-287, aa288-292, aa294-298) abolished the interaction between TBC-2 and RAB-10(Q68L). (D) Full-length TBC-2 interacts with AMPH-1. Mutation of key residues (prolines P150 or P153, or arginine R155) within TBC-2 (aa146-160), the predicted consensus sequence for AMPH-1 SH3-domain-binding, into alanine via site-directed mutagenesis disrupted the interaction between TBC-2 and the SH3 domain of AMPH-1. AMPH-1 SH3 domain was expressed as bait. Full-length and mutated forms of TBC-2 were expressed as prey. Their interactions were detected using the same two-hybrid assay as described in (A) and (B). (E) Schematic representation of TBC-2 domains and the truncated fragments of TBC-2 used in the Y2H analysis. Protein domains are displayed as dark boxes above the protein sequences (represented by dark lines). Amino acid numbers are indicated.
