STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis

ABSTRACT 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.

Y2H assay with negative controls: Empty vectors pGBKT7 (bait), a GAL4-DNA binding domain containing vector and pGADT7 (prey), a GAL4-activation domain containing vector were transformed into Y2HGold yeast strain. The obtained colonies were assayed for reporter activity and also tested for the expression of fusion proteins (Fig. S3B).
Y2H autoactivation assay: Y2HGold yeast strain was transformed with pGBKT7 vector containing wild-type or different mutants of STX13 and an empty pGADT7 vector. The obtained colonies were assayed for reporter activity and tested for the expression of fusion proteins (Fig. S3B). Similarly, Y2HGold yeast strain was transformed with an empty pGBKT7 vector and pGADT7 containing wild-type or different mutants of VAMP7 for testing the autoactivation of VAMP7 constructs ( Y2H interaction between STX13 and VAMP7: Y2HGold yeast strain was transformed with either pGBKT7 vector containing wild-type or Y3F mutant of STX13 and pGADT7 vector containing wild-type or different mutants of VAMP7. The obtained colonies were assayed on -His and -His containing 3AT (2mM) reporter activity plates and also tested for the expression of fusion proteins (Fig. S3D).
Y3H assay with negative controls: Empty vectors pBridge (bait), a GAL4-DNA binding domain containing vector and pGADT7 (prey), a GAL4-activation domain containing vector were transformed into Y2HGold yeast strain. The obtained colonies were assayed for reporter activity (Fig. 4F). J. Cell Sci. 128: doi:10.1242/jcs.171165: Supplementary Material Y3H autoactivation assay: Y2HGold yeast strain was transformed with pBridge vector containing either wild-type or Y3F mutant of STX13 at MCS I site and sigma3 or sigma1 at MCS II site and an empty pGADT7 vector. The obtained colonies were assayed for reporter activity (Fig. 4F) and tested for the expression of fusion proteins (Fig. S3C).

Journal of Cell Science | Supplementary Material
Y3H assay with positive controls, interaction between TYR and AP-3 or AP-1 hemicomplexes: Y2HGold yeast strain was transformed with pBridge vector containing TYR at MCS I site and sigma3 or sigma1 at MCS II site and pGADT7 vector containing AP-3 delta or AP-1 gamma subunits respectively. The obtained colonies were assayed for reporter activity (Fig. 4F) and tested for the expression of fusion proteins (Fig. S3C) (Sitaram et al., 2012).
Y3H interaction between STX13 and AP-3/AP-1 hemicomplexes: Y2HGold yeast strain was transformed with pBridge vector containing wild-type or Y3F mutant of STX13 at MCS I site and sigma3 or sigma1 at MCS II site and pGADT7 or pACT2 containing different subunits of AP-3 or AP-1 respectively. The obtained colonies were assayed for reporter activity (Fig. 4F) and tested for the expression of fusion proteins (Fig. S3C).  Immunoblotting (E) and immunofluorescence microscopy images (F) of STX13-depleted melanocytes before and after the treatment of bafilomycin A1. Arrowheads indicate the localization of TYR with respect to LAMP-2. Nucleus was stained with Hoechst 33258.

Journal of Cell Science | Supplementary Material
The insets are a magnified view of the white boxed areas. Scale bars, 10 m. J. Cell Sci. 128: doi:10.1242/jcs.171165: Supplementary Material

(B) A subset of melanosomal protein TYR localizes to STX13-positive structures.
Live cell imaging of GFP-TYR and RFP-STX13 in wild-type melanocytes. Arrows indicates the localization of TYR to STX13-postivie punctate or tubular compartments.

(C) Regulatory-domain-deficient STX13 mutants localizes to melanosomes.
Immunoelectron microscopy images of melanocytes stably expressing Myc-STX13 WT or mutants. Myc (PAG15) and TYRP1 (PAG10) represent the immunogold labeling of proteins with respective antibodies. Arrow and arrowheads represent the localization of STX13 to tubular endosomal domains and melanosomes (Stages III or IV) respectively.

assays. (B) STX13 deletion mutants but not VAMP7 showed autoactivation in Y2H
assay. Transformed the yeast strain Y2HGold with STX13 bait and empty prey plasmids or empty bait and VAMP7 prey plasmids as shown in the figure (also see Table S1) and selected the transformants on +His plates. For positive control, transformed the yeast cells with STX13 (WT or Y3F mutant) bait plasmid and pallidin (a BLOC-1 subunit) prey plasmid, and for negative control, transformed the yeast cells with empty bait and prey plasmids separately. Selected the transformants on +His, -His and -His (2 mM 3AT) reporter activity plates. Note that the STX13 mutants 129, 14-129 and 14-129 with Y3F showed autoactivation on -His (3AT) plates in the assay. Yeast transformants expressing respective bait and prey plasmids as shown in the figure were lysed using a protocol described in Materials and Methods and then subjected to immunoblotting. Blots were probed with anti-Myc or anti-HA antibodies. (C) Analysis of bait and prey protein expression in Y3H assay between STX13 and AP-3 or AP-1 hemicomplexes.
Y2HGold yeast strain expressing respective bait and prey plasmids as shown in the figure (also refer Fig. 4F) were lysed and then subjected to immunoblotting. Blots were probed with anti-STX13, anti-TYR, anti-Myc or anti-HA antibodies. Note that all subunits of AP-3 or AP-1 contain HA-epitope and STX13 and TYR were probed with their respective antibodies. Expression of empty vectors was also shown separately. (D) Y2H assay between STX13 and VAMP7, and the analysis of protein expression.
Transformed the yeast strain Y2HGold with STX13 (WT or Y3F mutant) bait and different VAMP7 prey plasmids as shown in the figure (also see Table S1). For reporter activity, selected the transformants on +His, -His and -His (2 mM 3AT) plates. Yeast transformants were lysed and then immunoblotted. Blots were probed with anti-HA or