Figure 3. Binding of either AP-3δ to... | Rockefeller University Press

Figure 3.

$Binding of either AP-3δ to VAMP7 or of Pldn to STX13 is required for pigmentation. (A and B) WT melan-Ink4a or Pldn-deficient melan-pa (Pldn−/−) cells stably expressing Myc-STX13 were transiently transfected to express GFP-VAMP7, -VAMP8, or -SEC22B. 1–2 d later, detergent cell lysates were prepared and immunoprecipitated using GFP-Trap agarose, and immunoprecipitates (IP) and 3% of the original cell lysates were fractionated by SDS-PAGE and analyzed by immunoblotting for STX13 or for GFP using HRP-conjugated secondary antibodies and analysis by ECL. (A) Representative images of relevant regions from the blots from a single experiment are shown. The upper band of the STX13 triplet represents Myc-STX13, and the two lower bands represent endogenous STX13. (B) Quantification from multiple experiments of coimmunoprecipitated STX13 band intensity (mean ± SEM) normalized to total STX13 in the lysate, immunoprecipitated GFP-tagged SNARE, and the value of STX13 coimmunoprecipitated (co-IP) with GFP-VAMP7; n = 3 (WT) or 4 (Pldn−/−). (C–H) The Bloc1s6 gene (encoding Pldn) in Ap3d1−/− melanocytes was disrupted by CRISPR/Cas9 mutagenesis to generate Ap3d1−/−/Pldn−/− cells. Data from a single clone and its transduced derivatives (representative of a second clone) are shown below. (C) RT-PCR of RNA from Ap3d1−/−/Pldn−/−, melan-pa (Pldn−/−), melan-pa cells stably expressing myc-tagged human Pldn (Pldn−/−:myc-Pldn), and melan-mh (Ap3d1−/−) cells, amplified for Pldn or tubulin-4B (as a loading control) were fractionated by agarose gel electrophoresis and visualized with SYBR Safe. Left: Positions of DNA markers (bp). Representative of three separate experiments. (D) Immunoblotting of fractionated melan-mh (Ap3d1−/−) and Ap3d1−/−/Pldn−/− whole-cell lysates for Pldn or γ-tubulin as a loading control. Left: Positions of molecular weight markers (kD). Representative of three separate experiments. (E and F) Fractionated whole-cell lysates of untransduced (−) melan-pa (Pldn−/−), melan-mh (Ap3d1−/−), or Ap3d1−/−/Pldn−/− cells or Ap3d1−/−/Pldn−/− cells transduced to express the indicated WT (black) or SNARE binding mutant (magenta) forms of AP-3δ and Pldn were immunoblotted for the BLOC-1 subunits Pldn and dysbindin (E), the AP-3 subunits δ and µ1 (F), or γ-tubulin (E and F). Left: Positions of molecular weight markers (kD). (G) Brightfield microscopy of untransduced Ap3d1−/−/Pldn−/− melanocytes (−) or cells expressing the indicated WT or SNARE binding mutant forms of AP-3δ and Pldn. Scale bar, 10 µm. (H) Melanin content in lysates of untransduced Ap3d1−/−/Pldn−/− cells (−) or cells expressing the indicated WT or SNARE binding mutant forms of AP-3δ and Pldn was assessed by spectrophotometry. Data (mean ± SEM from three separate experiments) are normalized to the mean value from cells expressing AP-3δWT and PldnWT. ***, P < 0.001; ****, P < 0.0001; nonsignificant differences are not indicated.$

Binding of either AP-3δ to VAMP7 or of Pldn to STX13 is required for pigmentation. (A and B) WT melan-Ink4a or Pldn-deficient melan-pa (Pldn^−/−) cells stably expressing Myc-STX13 were transiently transfected to express GFP-VAMP7, -VAMP8, or -SEC22B. 1–2 d later, detergent cell lysates were prepared and immunoprecipitated using GFP-Trap agarose, and immunoprecipitates (IP) and 3% of the original cell lysates were fractionated by SDS-PAGE and analyzed by immunoblotting for STX13 or for GFP using HRP-conjugated secondary antibodies and analysis by ECL. (A) Representative images of relevant regions from the blots from a single experiment are shown. The upper band of the STX13 triplet represents Myc-STX13, and the two lower bands represent endogenous STX13. (B) Quantification from multiple experiments of coimmunoprecipitated STX13 band intensity (mean ± SEM) normalized to total STX13 in the lysate, immunoprecipitated GFP-tagged SNARE, and the value of STX13 coimmunoprecipitated (co-IP) with GFP-VAMP7; n = 3 (WT) or 4 (Pldn^−/−). (C–H) The Bloc1s6 gene (encoding Pldn) in Ap3d1^−/− melanocytes was disrupted by CRISPR/Cas9 mutagenesis to generate Ap3d1^−/−/Pldn^−/− cells. Data from a single clone and its transduced derivatives (representative of a second clone) are shown below. (C) RT-PCR of RNA from Ap3d1^−/−/Pldn^−/−, melan-pa (Pldn^−/−), melan-pa cells stably expressing myc-tagged human Pldn (Pldn^−/−:myc-Pldn), and melan-mh (Ap3d1^−/−) cells, amplified for Pldn or tubulin-4B (as a loading control) were fractionated by agarose gel electrophoresis and visualized with SYBR Safe. Left: Positions of DNA markers (bp). Representative of three separate experiments. (D) Immunoblotting of fractionated melan-mh (Ap3d1^−/−) and Ap3d1^−/−/Pldn^−/− whole-cell lysates for Pldn or γ-tubulin as a loading control. Left: Positions of molecular weight markers (kD). Representative of three separate experiments. (E and F) Fractionated whole-cell lysates of untransduced (−) melan-pa (Pldn^−/−), melan-mh (Ap3d1^−/−), or Ap3d1^−/−/Pldn^−/− cells or Ap3d1^−/−/Pldn^−/− cells transduced to express the indicated WT (black) or SNARE binding mutant (magenta) forms of AP-3δ and Pldn were immunoblotted for the BLOC-1 subunits Pldn and dysbindin (E), the AP-3 subunits δ and µ1 (F), or γ-tubulin (E and F). Left: Positions of molecular weight markers (kD). (G) Brightfield microscopy of untransduced Ap3d1^−/−/Pldn^−/− melanocytes (−) or cells expressing the indicated WT or SNARE binding mutant forms of AP-3δ and Pldn. Scale bar, 10 µm. (H) Melanin content in lysates of untransduced Ap3d1^−/−/Pldn^−/− cells (−) or cells expressing the indicated WT or SNARE binding mutant forms of AP-3δ and Pldn was assessed by spectrophotometry. Data (mean ± SEM from three separate experiments) are normalized to the mean value from cells expressing AP-3δ^WT and Pldn^WT. ***, P < 0.001; ****, P < 0.0001; nonsignificant differences are not indicated.