Figure 1.

A) Rab31 interacts with OCRL-1. (I) Y187 yeast strain transformed with the BD/OCRL-1 plasmid was mated with the AH109 yeast strain transformed with the following plasmid: a) AD/Rab31, b) AD/unrelated protein, c) AD/empty. (II) Y187 strain transformed with AD/Rab31 was mated with the AH109 strain transformed with following plasmid: d) BD/unrelated protein and e) BD/empty plasmid. The mating mixtures were first plated on agar containing double drop out (DO) media (1), to assess for mating efficiency. Then, a replica plate onto agar containing quadrate drop out media (QO, high stringency media) was made (2), to identify the diploids expressing fusion proteins that interact. Additionally, the expression of the lacZ gene was assessed; X-α-gal was used as α-galactosidase substrate (interaction results in growth of blue colonies). B) Interaction of OCRL-1 with Rab proteins. The capacity of OCRL-1 to interact with other Rab proteins was assessed using the yeast two hybrid system. The AH109 yeast strain transformed with BD/OCRL-1 plasmid was mated with the Y187 yeast strain transformed with the following plasmid: a) AD/rRab22b, b) AD/ Rab1, c) AD/Rab5, d)AD/Rab8, e) AD/Rab14 and f) AD/Rab40c g) positive control and h) negative control. C) Formation of OCRL-1-Rab31 complex does not depend on the conformational state of Rab31. (I) The AH109 yeast strain transformed with BD/OCRL-1 plasmid was mated with the Y187 yeast strain transformed with the following plasmid: a) AD/Rab31, b) AD/ Rab31(S19N), c) AD/Rab31(Q64L). (II) Positive control and negative control. Positive experimental controls were carried out by mating AH109 host strain transfected with pGBKT7-53 and Y187 transfected with pGADT7-T. pGBKT7-53 encode fusions between the GAL4 DNA-BD and murine p53, while pGBKT7-T -encode fusions between AD and SV40 large T-antigen. Negative experimental controls were carried out by mating AH109 host strain transfected with pGBKT7-Lam with Y187 transfected with pGADT7-T. pGBKT7-Lam encode a fusion of the DNA-BD with human lamin C. D) Mapping of the Rab31 binding site in OCRL-1. Full-length and truncated OCRL-1 constructs were tested for interaction with Rab31 using the yeast two-hybrid system. The Y187 yeast strain transformed with AD/Rab31 plasmid was mated with the AH109 yeast strain transformed with the following plasmids: a) BD/OCRL-1, b) BD/Δ (1-539) OCRL-1, c) BD/Δ(1-559) OCRL-1, d) BD/Δ(540-559) OCRL-1, e) BD/Δ (650-900) OCRL-1 and f) BD/Δ (858-900) OCRL-1. E) GST-Rab31 pull-down experiments. [³⁵S]-labeled OCRL-1 protein was incubated in a media containing 100 μM GTP with either GST-Rab31 (I) or GST (II). The GST-Rab31-OCRL-1 complex was pulled down with agarose-glutathione beads. The proteins were separated by SDS-PAGE. The band containing the [³⁵S]-labeled protein was revealed using a fluographic reagent and exposing the gel overnight to X-ray film. Number to the left indicates the molecular weight of [³⁵S]-labeled OCRL-1 protein.