Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Abstract

Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.

Keywords: AP-3; BLOC-1; BLOC-2; BLOC-3; HPS; Rab38/Rab32; Rab9A.

Figure 1

Localization and function of Rab9A in wild-type melanocytes. (A–E, G) Rab9A^WT and Rab9A^Q66L localizes primarily to lysosomes, with a subset in melanosomes and endosomes. Wild-type melanocytes were transiently transfected with 1 μg of GFP-Rab9A^WT or GFP-Rab9A^Q66L and then analyzed by BF and IFM. Arrows point to tubular GFP-Rab9A structures that are colocalized or associated with LAMP-2 (A) or EEA1 (C) or STX13 (D), in proximity to TYPR1 (B) or associated with BF melanosomes (pseudocoloured to blue) (A-D). Arrowheads point to the ring-like or punctate GFP-Rab9A (WT or Q66L) structures that are colocalized with LAMP-2 and melanosomes (pseudocoloured to blue) (A, G), TYRP1 (B, G), EEA1 (C, G), or STX13 (D). Graph (E) represents the colocalization efficiency between GFP-Rab9A (WT or Q66L) and other markers measured as Pearson’s correlation coefficient (r, n = ∼5–7 cells). Values in parenthesis indicate the colocalization coefficient between the two marker proteins. (F) Tubular GFP-Rab9A^WT structures associated with melanosomes in live imaging microscopy. Cells were transfected with 1 μg of GFP-Rab9A and then imaged by live imaging microscopy. Insets represent GFP-Rab9A localization alone (top panel) or relative to melanosomes (bottom panel) at different time points. Arrows point to the tubular GFP-Rab9A structures. (H) Rab9A^S22N localizes to cytosol in melanocytes. Wild-type melanocytes were transiently transfected with 1 μg of GFP-Rab9A^S22N and then analyzed by BF and IFM. (I) Overexpression of Rab9A increases the melanocyte pigmentation. Wild-type melanocytes were transiently transfected with 2 μg of different isoform of GFP-Rab9A or empty vector (as a control) and then measured the melanin pigments (n = 2). Values indicate the fold change in pigmentation relative to control.

Figure 2

ShRNA-mediated depletion of Rab9A in wild-type melanocytes. (A) Rab9A knockdown results in hypopigmentation, similar to BLOC-3⁻ cells or Rab38/32 or VARP-depleted melanocytes. Cells were transduced with retrovirus encoding shRNA against RAB9A, RAB38, RAB32, or VARP genes and analyzed by BFM. (B and C) Rab9A depletion affects the melanocyte pigmentation, similar to Rab38/32 or VARP-knockdown or Rab9A-38 or Rab9A-32 double knockdown. Wild-type melanocytes were transduced with retrovirus encoding respective shRNA as labeled and measured the melanin pigments (n = 2). BLOC-3⁻ cells are mouse melanocytes deficient for HPS4 subunit. Values indicate the fold change in pigmentation relative to control. (D) Localization of GFP-Rab9A to lysosomes increases upon knockdown of Rab38/32 or VARP, or in BLOC-1⁻, −3⁻, or AP-3⁻ melanocytes. Cells were transiently transfected with 2 μg of GFP-Rab9A and then analyzed by BF and IFM. Arrowheads point to tubular or ring-like GFP-Rab9A structures that are positive for LAMP-2 in all cells and closely associated or colocalized with hypopigmented melanosomes (pseudocoloured to blue) in knockdown cells. Note that GFP-Rab9A localization to LAMP-2-positive organelles is significantly decreased in BLOC-2⁻ cells. Nuclei were stained with Hoechst. Bars, 10 μm and insets, 2.5X of white boxed regions.

Figure 3

Localization and steady-state levels of Rab38 in Rab9A-knockdown cells. (A, C) HA-Rab38 localizes to melanosomes in wild type and BLOC-2-deficient cells. Its localization to recycling endosomes increases upon BLOC-3-deficiency or knockdown of Rab9A, Rab32 or VARP in wild-type melanocytes. Cells were transiently transfected with 2 μg of HA-Rab38 and analyzed by BF and IFM. Arrowheads point to the Rab38 localization with respect to melanosomes (pseudocoloured to blue) or endosomal STX13. Graph (C) represents the colocalization efficiency between HA-Rab38 and STX13 measured as Pearson’s correlation coefficient (r, n = 5 cells). Values in parenthesis indicate the colocalization coefficient between HA-Rab38 and STX13. &, P < 0.05; &&, P < 0.01 and &&&, P < 0.001; mean±s.e.m. (B) Localization of HA-Rab38 to early endosomes increases upon Rab9A-depletion in wild-type melanocytes. Rab9A-knockdown or control cells were transfected with HA-Rab38 and analyzed by BF and IFM. Arrowheads point to the Rab38 localization with respect to melanosomes (pseudocoloured to blue) or endosomal EEA1 or Rab5. Values in parenthesis indicate the colocalization coefficient between HA-Rab38 and EEA1 or Rab5. Nuclei were stained with Hoechst. Bars, 10 μm and insets, 2.5X of white boxed regions. (D) Rab9 expression is slightly but Rab38 expression significantly reduced in Rab9A, Rab38/32 or VARP-knockdown or BLOC-3⁻ melanocytes. Immunoblot analysis of cellular Rab9 and Rab38 protein levels, with γ-Tubulin as a loading control. Values in parenthesis indicate the fold change in band intensity relative to control. Note that the band intensities were normalized with their respective tubulin prior to the fold change calculation.

Figure 4

Steady-state distribution of melanosomal proteins and their expression in Rab9A-depleted melanocytes. (A, B) Rab9A knockdown leads to mislocalization of melanosome cargo to lysosomes, similar to the phenotype observed in BLOC-3⁻, or Rab38/32, or VARP-knockdown melanocytes. Cells were fixed, stained for melanosomal proteins and analyzed by BF and IFM. Arrowheads point to the melanosomal proteins TYRP1, TYR, and PMEL and their localization relative to either the lysosomal protein LAMP-2 or melanosomes (pseudocoloured to blue). Arrows point to the localization of cargo to melanosomes that are colocalized or associated with LAMP-2 in BLOC-3-deficient melanocytes. Nuclei were stained with Hoechst. Bars, 10 μm and insets, 2.5X of white boxed regions. Graph (B) represents the colocalization efficiency between TYRP1 or GFP-TYR or PMEL and LAMP2 measured as Pearson’s correlation coefficient (r, n = 5 cells). Values in parenthesis indicate the colocalization coefficient between the two marker proteins. ns, not significant; &, P < 0.05; &&, P < 0.01 and &&&, P < 0.001; mean ± SEM. (C) Melanosomal protein expression is dramatically reduced in Rab9A, Rab38/32 or VARP-knockdown or BLOC-3-deficient melanocytes. Immunoblot analysis of melanosomal proteins TYRP1 and TYR, with γ-Tubulin as a loading control. Values in parenthesis indicate the fold change in band intensity relative to control. Note that the band intensities were normalized with their respective tubulin prior to the fold change calculation.

Figure 5

Live imaging of tubular recycling structures and their cellular distribution in Rab9A, Rab38/32, and VARP-knockdown or BLOC-3-deficient melanocytes. (A) Rab9A, Rab38/32 or VARP-knockdown or BLOC-3-deficiency in melanocytes alters the length of STX13-positive recycling endosomes. Gene knockdown or HPS-deficient melanocytes were transiently transfected with 2 μg of GFP-STX13 and then imaged by live microscopy. Insets represent GFP localization at different time points, and their respective skeleton images are shown separately. Arrows point to tubular GFP-STX13 structures. (B, C) Rab9A, Rab38/32 or VARP-knockdown or BLOC-3-deficient cells mislocalize the endosomal recycling SNARE, GFP-STX13, and melanosomal protein TYRP1 to lysosomes. Cells were transiently transfected with 2 μg of GFP-STX13, fixed, counter-stained with either TYRP1 or LAMP-2 and then analyzed by BF and IFM (C). Arrowheads point to GFP-STX13 localization. The colocalization efficiency between GFP-STX13 and TYRP1 or LAMP-2 was measured as Pearson’s correlation coefficient (r, n = ∼5–6 cells) and then plotted (B). (B, D) Rab9A or Rab38/32-depletion in melanocytes leads to partial mislocalization of endogenous STX13 to lysosomes. Knockdown melanocytes were fixed, stained for either the early endosomal marker EEA1 or the lysosomal marker LAMP-2, and then analyzed by IFM (D). Arrowheads point to STX13 localization. The colocalization efficiency between STX13 and EEA1 or LAMP-2 was measured as Pearson’s correlation coefficient (r, n = ∼5–6 cells) and then plotted (B). Values in parenthesis indicate the colocalization coefficient between the two marker proteins. Statistical significance in r value between control sh and Rab9A sh, Rab38/32 sh, VARP sh, or BLOC-3⁻ cells was measured using graphpad software. ns, not significant; &&, P < 0.01 and &&&, P < 0.001; mean±s.e.m. Nuclei were stained with Hoechst. Bars, 10 μm and insets, 2.5X of white boxed regions. (E) STX13 expression is significantly reduced in Rab9A, Rab38/32 or VARP-knockdown or BLOC-3-deficient melanocytes. Immunoblot analysis of cellular STX13 levels with γ-Tubulin as a loading control. Protein band intensities were quantified and normalized to γ-Tubulin. The fold change relative to control cells was then calculated as described in Supporting Information. Values in parenthesis indicate the fold change in band intensity relative to control. Note that the band intensities were normalized with their respective tubulin prior to the fold change calculation.

Figure 6

Model depicting the function of Rab9A and its co-regulators and HPS complexes in melanosome cargo transport and pigmentation. (A) Cellular pigmentation and localization of melanosomal cargo and endosomal SNARE in the shRNA-depleted or HPS mutant melanocytes. Cells were fixed, stained, and analyzed by IFM. Melanocyte pigmentation was analyzed by BFM. Organelle-specific markers were used to study the distribution of cargo or STX13 in all cells. Red text indicates a subset of cargo or SNARE localization to the indicated organelles. Mel., Melanosomes; Pre-mel., stage II melanosomes; Endo., endosomes; Lyso., lysosomes; PM, plasma membrane; Dark, normal pigmentation; and Hypo, hypopigmentation. Note that the localization of melanosomal cargo in BLOC-1⁻, BLOC-2⁻, and AP-3⁻ cells was characterized extensively in (Dennis et al., ; Setty et al., ; Theos et al., 2005). (B) Schematic representation of cargo transport from recycling endosomes to melanosome through STX13-mediated membrane fusion. Model representing the cargo TYRP1 (BLOC-1-dependent cargo, orange symbols) and TYR (AP-3-dependent cargo, blue symbols) transport from tubular-vesicular structures of recycling endosomes during melanosome biogenesis. STX13 (Qa SNARE, green) with two other unknown SNAREs (Qb, Qc; black) on recycling endosomes makes trans-SNARE complex with VAMP7 (R-SNARE, black) on Stage III or Stage II (not shown) melanosome during cargo delivery. Upon cargo transport and melanin synthesis, Stage III matures into Stage IV melanosome. Curved arrows represent the fusion of endosomal transport carriers with melanosomes. Dotted arrows point to the mislocalization of cargo to other organelles in the absence or deficiency (represented as inhibition symbol, red) of respective molecules or complexes. The solid black lines in the stage III melanosome represent the PMEL fibrils. Based on the data presented here and previous literature, BLOC-1 and AP-3 possibly function at early and BLOC-2/Rab9A/BLOC-3/Rab38/32/VARP later stages of cargo transport to the melanosome.