Genetic analyses of Drosophila homologs of non-canonical BLOC-1 subunits

BLOC-1 (biogenesis of lysosome-related organelles complex 1) is a eukaryotic protein complex required for the efficient formation of lysosome related organelles (LROs), a class of membrane-bound organelles derived from the endosomal-lysosomal pathway. In metazoans, BLOC-1 is an octameric protein complex consisting of pallidin, muted, dysbindin, cappuccino, snapin, BLOS1, BLOS2 and BLOS3. Mutation of the genes encoding dysbindin, pallidin or BLOS3 causes Hermansky-Pudlak syndrome in humans, and mutation of the genes encoding pallidin, muted, dysbindin, cappuccino or BLOS3 causes a related syndrome in mice. To genetically determine the role of Drosophila homologs of non-canonical BLOC-1 proteins in relation to canonical BLOC-1, I deleted a non-canonical BLOC-1 gene by P element imprecise excision and a second non-canonical BLOC-1 gene by CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated protein 9)-mediated genomic editing. At the same time I deleted Drosophila Blos3, also using a CRISPR-based approach. Blos3 is unique among the eight metazoan BLOC-1 subunit genes as mutation of Blos3 causes an atypically mild BLOC-1 phenotype in mice. I found that deletion of one of the non-canonical BLOC-1 proteins did not affect the biogenesis of eye pigment granules in Drosophila, which are LROs that require BLOC 1 function for normal biogenesis; nor did it modify function of BLOC-2, a protein complex previously shown to act epistatically to BLOC-1. On the other hand, deletion of the second non-canonical BLOC-1 protein resulted in higher eye pigment levels than those of wildtype flies. Lastly, I found that deletion of Blos3 caused attenuated BLOC-1 phenotypes in Drosophila. These findings support the interpretation that the two non-canonical BLOC-1 proteins that I deleted do not have roles in canonical BLOC-1 activity, though one of them may negatively regulate BLOC-1 function. Finally Blos3 appears to function in a unique manner among the recognized BLOC-1 subunits even across distantly related eukaryotic species