Rab22A recruits BLOC‐1 and BLOC‐2 to promote the biogenesis of recycling endosomes

Abstract Recycling endosomes (REs) are transient endosomal tubular intermediates of early/sorting endosomes (E/SEs) that function in cargo recycling to the cell surface and deliver the cell type‐specific cargo to lysosome‐related organelles such as melanosomes in melanocytes. However, the mechanism of RE biogenesis is largely unknown. In this study, by using an endosomal Rab‐specific RNAi screen, we identified Rab22A as a critical player during RE biogenesis. Rab22A‐knockdown results in reduced RE dynamics and concurrent cargo accumulation in the E/SEs or lysosomes. Rab22A forms a complex with BLOC‐1, BLOC‐2 and the kinesin‐3 family motor KIF13A on endosomes. Consistently, the RE‐dependent transport defects observed in Rab22A‐depleted cells phenocopy those in BLOC‐1‐/BLOC‐2‐deficient cells. Further, Rab22A depletion reduced the membrane association of BLOC‐1/BLOC‐2. Taken together, these findings suggest that Rab22A promotes the assembly of a BLOC‐1‐BLOC‐2‐KIF13A complex on E/SEs to generate REs that maintain cellular and organelle homeostasis.

. Analysis of endosomal Rab RNAi screen and the localization of Rabs to REs.
A IFM images of HeLa cells to study the localization of KIF13A-YFP to REs. B PCR analysis of Rab-knockdown HeLa cells to measure the knockdown efficiency of each shRNA as indicated. DNA band intensities were quantified and indicated on the gels. C Subcellular fractionation of HeLa cells to study the localization of different Rabs (red box) with respect to STX13 or AP-1. D, E IFM analysis of HeLa cells that were transfected with different constructs as indicated.   Figure EV2. Analysis of mouse melanocytes expressing different constructs of Rab22A.
A Live cell imaging of mCherry-Rab22A with respect to GFP-STX13 in wild-type melanocytes. Arrows point to Rab22A-positive buds or tubular structures arising from STX13-positive E/SEs (arrowheads). Magnified view of insets (at various seconds) is shown separately. Scale bars: 10 lm. B IFM analysis of mCherry-Rab22A WT/Q64L/S19N -transfected melanocytes. Arrowheads point to Rab22A localization, and arrows indicate the melanocyte pigmentation.
Nuclei are stained with Hoechst 33258. The colocalization coefficient (r, in mean AE SEM) between two markers was indicated separately. Scale bars: 10 lm. C, D Quantification of melanin content (C, n = 5) and the protein levels of TYRP1 (judged by immunoblotting in D, n = 3) in melanocytes that were transfected with GFP-Rab22A WT/Q64L/S19N . In (D), graph indicates the relative levels (mean AE SEM) of TYRP1 with respect to overexpressed Rab22A. Protein band intensities were quantified and indicated on the gels. **P ≤ 0.01, ***P ≤ 0.001 and ns = not significant (unpaired Student's t-test).
A IFM images of KIF13A-YFP-transfected control and knockdown HeLa cells as indicated. Arrowheads point to the localization of KIF13A to E/SEs or REs. B Membrane-cytosol fractionation of HeLa cell homogenate for the localization of KIF13A. *, non-specific bands. C IFM images of control and Rab22A-, BLOC-1-knockdown HeLa cells that were stained with LAMP-2 or internalized with fluorescein-dextran. D Cell surface levels of LAMP-1 and M6PR in control and Rab22A-, BLOC-1-knockdown HeLa cells measured using flow cytometry. Normalized mean fluorescence intensity (MFI) was calculated (mean AE SEM) and then plotted. n = 3. E IFM images of HeLa cells that were subjected to Tf-Alexa Fluor 594 recycling kinetics. F Fluorescence intensities Tf in the images of (E) were quantified and plotted (mean AE SEM). n = 3. n c = total number of cells. G Immunoblotting analysis of Tf receptor in HeLa cells as indicated. Protein band intensities were quantified and indicated on the gels.
Data information: In (A, C, E), arrows point to the localization of cytoskeletal proteins (A) or internalized dextran or lysosomes (C) or accumulation of Tf to the intracellular vesicles (E). Scale bars: 10 lm. In (D, F), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ns = not significant (unpaired Student's t-test). PCR analysis of Rab22A-depleted melanocytes. DNA band intensities were quantified and indicated on the gels. C BF and IFM images of GFP-Rab22A WT/Q64L/S19N -transfected BLOC-1-deficient melanocytes. Black and white arrows indicate the melanocyte pigmentation and GFP-Rab22A expression, respectively. Scale bars: 10 lm. D Subcellular membrane fractionation of control and knockdown HeLa cells. The fractions were probed for HPS6 (red box). E, F Graphs represent the percentage of membrane association of pallidin or HPS6 or Rab22A in the indicated cell types and derived from Fig 5B and C, respectively. n = 1. G Pull-down of His-Rab22A WT/Q64L using melanocyte lysate. The bead-bound His-Rab22A was shown as coomassie-stained gel separately. H Transcript levels of Rab22A and pallidin (BLOC-1) in control and schizophrenia patients. Analysed from data set GSE53987 and then plotted (mean AE SEM). n = 15-18 samples. *P ≤ 0.05 and **P ≤ 0.01 (unpaired Student's t-test).