Sec8 and Myo15 regulate epidermal chitin synthase localization and cuticular chitin biosynthesis

Chitin synthase 1 (Chs1) is an integral membrane β-1,4-glycosyltransferase that catalyzes chitin biosynthesis, which is essential for insect cuticle formation. Proper localization of Chs1 to the apical plasma membrane of epidermal cells is critical for chitin deposition in the cuticle; however, the mechanisms regulating this localization remain unclear. Here, using TurboID-based proximity biotinylation of epidermal Chs1 (Krotzkopf verkehrt, Kkv) in Drosophila melanogaster, we established a Kkv proximal proteome and identified two candidate interacting proteins, DmSec8 and DmMyo15. Split-ubiquitin membrane yeast two-hybrid and pull-down assays confirmed the physical interactions between Kkv and these proteins. RNA interference (RNAi) of the corresponding homologs in Tribolium castaneum revealed that silencing either Sec8 or Myo15 resulted in lethality, molting defects, reduced cuticular chitin levels, and mislocalization of Chs1 from the apical membrane to perinuclear regions. Together, these findings identify Sec8 and Myo15 as previously unrecognized regulators of Chs1 membrane localization, providing new insights into the molecular mechanisms underlying chitin biosynthesis in insects.