Fig. 3: LRMDA interacts with RAB32 through N-terminal leucine rich repeats and acts as a linker between RAB32 and Retriever. | Nature Communications

Fig. 3: LRMDA interacts with RAB32 through N-terminal leucine rich repeats and acts as a linker between RAB32 and Retriever.

From: Identification of a RAB32-LRMDA-Commander membrane trafficking complex reveals the molecular mechanism of human oculocutaneous albinism type 7

Fig. 3

A AlphaFold2 model showing the assembly of LRMDA and RAB32, with top panel highlighting the interfacial residues (B, C) and bottom panel showing the position of RAB32 T39 and Q85 residues that were substituted to generate constitutively inactive and active RAB32 mutants, respectively (D). B HEK293T cells were transfected with GFP, GFP-LRMDA or mutants of the interfacial residues to probe RAB32 interaction: GFP-LRMDA Y106D, L119D, F145D. The lysates were used in GFP-trap experiments to analyze the association with the Retriever complex or RAB32. n = 3 biological replicates, 2-way ANOVA with Dunnett’s multiple comparison test, data presented as mean values and error bars represent s.d.; n.s., denotes changes with p > 0.05. C HEK293T cells were transfected with GFP, GFP-RAB32 or mutants of the interfacial residues to probe LRMDA interaction: GFP-RAB32 D61L, F62D, L64D. The lysates were used in GFP-trap experiments to analyze the association with Retriever complex or LRMDA. n = 3 biological replicates, 2-way ANOVA with Dunnett’s multiple comparison test, data presented as mean values and error bars represent s.d. D HEK293T cells were transfected with GFP, GFP-RAB32 or constitutively active GFP-RAB32 Q85L or inactive T39N. The lysates were used in GFP-trap experiments to analyze the association with Retriever complex or LRMDA. n = 3 biological replicates, 2-way ANOVA with Dunnett’s multiple comparison test, data presented as mean values and error bars represent s.d.; n.s. denotes changes with p > 0.05. E Recombinantly purified Retriever, containing His-VPS29, was bound to TALON resin. The bead-bound Retriever was mixed with recombinantly purified LRMDA, RAB32 or LRMDA and RAB32. The normalised band intensities were compared to samples with added RAB32 and LRMDA. n = 3 biological replicates, 2-way ANOVA with Dunnett’s multiple comparison test, data presented as mean values and error bars represent s.d.

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