STX13 binding by the BLOC-1 subunit Pldn is not required for pigmentation or melanosome cargo localization in melanocytes. (A) Yeast two-hybrid assay for WT or triple alanine scanning mutants of full-length Pldn (fused to the GAL4 activation domain) binding to either the STX13 cytoplasmic domain or full-length Pldn (fused to the GAL4 DNA binding domain). A truncated Pldn mutant harboring residues 60–100 and a full-length mutant with residues 110–124 mutagenized to alanines (110-124A) are negative controls. Representative of at least two experiments showing growth of transformed his3 yeast colonies harboring HIS3 driven by the GAL4 promoter on plates lacking (−His) or containing (+His) histidine; growth on −His plates indicates an interaction. (B–F) WT Pldn (Pldn^WT) or the indicated mutants were expressed stably in Pldn-deficient melan-pa (Pldn^−/−) melanocytes by retroviral transduction. (B) Melanin content in cell lysates was quantified by spectrophotometry. Data (mean ± SEM from two separate experiments done in triplicate) are normalized to the mean value from cells expressing Pldn^WT. *, P < 0.05; **. P < 0.01; if not indicated, values were not significantly different from cells expressing Pldn^WT. (C–F) Untransduced melan-pa (Pldn^−/−) melanocytes (C) or stable transductants expressing Pldn^WT (D), a mutant that does not bind STX13 (Pldn^110-112A; E) or a mutant that retains STX13 binding (Pldn^164-166A; F) were fixed, immunolabeled for TYRP1 and STX13, and analyzed by dIFM and brightfield microscopy. Boxed regions are magnified fivefold in insets. White arrows, TYRP1 colocalized with STX13; cyan arrows, TYRP1 on melanosomes; arrowheads, STX13 on TYRP1-negative compartments. Scale bar, 10 µm; inset scale bar, 2 µm.