Fig. 4: The direct coupling between RAB32-LRMDA-Retriever is important for human pigmentation. | Nature Communications

Fig. 4: The direct coupling between RAB32-LRMDA-Retriever is important for human pigmentation.

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

Fig. 4

A, B Volcano plot showing the loss of proteins in GFP-LRMDA F145D (A) or GFP-LRMDA L226G (B) pulldown in RPE1 cells, compared to wild-type GFP-LRMDA sample. n = 6, two-tailed paired t-test, normalised to LRMDA levels. Significantly decreased proteins with p < 0.01 and Log2 fold change < −2 are labelled. Proteins with FDR < 0.05 are depicted with triangles. C Schematic depicting the position of reported OCA7-causative mutations in LRMDA. The numbering for the longer LRMDA isoform (Uniprot: A0A087WWI0) is listed in black font, and the shorter isoform (UniProt: Q9H2I8) is listed in grey. D Mapping of OCA7-causative mutations in LRMDA onto AlphaFold2 model of RAB32-LRMDA-Retriever assembly. E HEK293T cells were transfected with GFP, GFP-LRMDA or OCA7-causative mutations: GFP-LRMDA N117K, Y208stop, G217S or R222stop. The lysates were used in GFP-trap experiments to analyze the association with the Retriever complex or RAB32. n = 3 biological replicates, 1-way ANOVA with Dunnett’s multiple comparison test, data presented as mean values and error bars represent s.d. F RAB32 KO in MNT1 cells validated using western blotting. n = 3 biological replicates, 1-way ANOVA with Dunnett’s multiple comparison test, data presented as mean values and error bars represent s.d. G Cell pellets of different RAB32 KO clones, showing a minor decrease in pigmentation. H Measurement of RAB32 KO melanin levels compared to parental MNT1 cell line. n = 3 biological replicates, 1-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. I LRMDA KO MNT1 cells were lentivirally transduced with GFP, wild-type GFP-LRMDA or mutants GFP-LRMDA F145D and L226G, that perturb binding with RAB32 or Retriever, respectively. Data representative of 2 independent repeats. J Cell pellets of LRMDA KO cells and cells lentivirally rescued with GFP, wild-type GFP-LRMDA or mutants GFP-LRMDA F145D and L226G, showing a difference in pigmentation. K The pellets from (J) were used for melanin content measurements. n = 3 biological replicates, 1-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.

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