Melanosomes are a class of lysosome-related organelles produced by melanocytes. Biogenesis of melanosomes requires the transport of melanin-synthesizing enzymes from tubular recycling endosomes to maturing melanosomes. The SNARE proteins involved in these transport or fusion steps have been poorly studied. We found that depletion of syntaxin 13 (STX13, also known as STX12), a recycling endosomal Qa-SNARE, inhibits pigment granule maturation in melanocytes by rerouting the melanosomal proteins such as TYR and TYRP1 to lysosomes. Furthermore, live-cell imaging and electron microscopy studies showed that STX13 co-distributed with melanosomal cargo in the tubular-vesicular endosomes that are closely associated with the maturing melanosomes. STX family proteins contain an N-terminal regulatory domain, and deletion of this domain in STX13 increases both the SNARE activity in vivo and melanosome cargo transport and pigmentation, suggesting that STX13 acts as a fusion SNARE in melanosomal trafficking pathways. In addition, STX13-dependent cargo transport requires the melanosomal R-SNARE VAMP7, and its silencing blocks the melanosome maturation, reflecting a defect in endosome–melanosome fusion. Moreover, we show mutual dependency between STX13 and VAMP7 in regulating their localization for efficient cargo delivery to melanosomes.

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.

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.