Motif‐guided trafficking of leishmanial <scp>PAS</scp> domain‐containing phosphoglycerate kinase into glycosomes and lysosomes

Recently, we showed that the Per-ARNT-Sim (PAS) domain-containing phosphoglycerate kinase from Leishmania major (LmPAS-PGK) is imported into both the glycosome and the lysosome; however, the mechanism underlying its dual targeting has remained unclear. LmPAS-PGK contains a C-terminal tripeptide sequence and two dileucine base motifs. To investigate the roles of these sorting signals, we generated different complement cell lines from null mutants by transfecting constructs encoding the wild-type protein, an L230A/L231A mutant, the C-terminal tripeptide deleted variant (∆^525-527), a dileucine motif deleted variant (∆^504-508), and a double-deletion mutant (∆^525-527/∆^504-508). Our results demonstrate that the dileucine motif governs the lysosomal targeting of LmPAS-PGK, whereas the C-terminal tripeptide is required for glycosomal localization. Deletion of both motifs abolished trafficking to either organelle, leading to cytosolic redistribution. Notably, ∆^525-527, ∆^504-508, and ∆^525-527/∆^504-508 cells displayed lower virulence compared with control cells in infected macrophages, underscoring the importance of proper LmPAS-PGK localization in Leishmania pathogenicity. Impact statement Here, we show the dileucine motif of PAS-PGK of Leishmania major governs its lysosomal targeting, whereas its C-terminal tripeptide is required for glycosomal localization. Cells lacking these domains displayed lower virulence compared with control cells in infected macrophages. These results increase our understanding of intracellular trafficking, as well as host-parasite interactions.