Abstract
The limiting membrane of lysosomes in animal cells and that of the vacuole in yeast include a wide variety of transporters, but little is known about how these proteins reach their destination membrane. The mammalian PQLC2 protein catalyzes efflux of basic amino acids from the lysosome, and the similar Ypq1, −2, and −3 proteins of yeast perform an equivalent function at the vacuole. We here show that the Ypq proteins are delivered to the vacuolar membrane via the alkaline phosphatase (ALP) trafficking pathway, which requires the AP-3 adaptor complex. When traffic via this pathway is deficient, the Ypq proteins pass through endosomes from where Ypq1 and Ypq2 properly reach the vacuolar membrane whereas Ypq3 is missorted to the vacuolar lumen via the multivesicular body pathway. When produced in yeast, PQLC2 also reaches the vacuolar membrane via the ALP pathway, but tends to sort to the vacuolar lumen if AP-3 is defective. Finally, in HeLa cells, inhibiting the synthesis of an AP-3 subunit also impairs sorting of PQLC2 to lysosomes. Our results suggest the existence of a conserved AP-3-dependent trafficking pathway for proper delivery of basic amino acid exporters to the yeast vacuole and to lysosomes of human cells.
Highlights
These transporters is a triple helix bundle (THB)[10]
In the vps27Δ mutant, PQLC2LL>AA was stacked in a large perivacuolar compartment: the class E compartment typically observed in this category of mutant (Fig. 6B). These results show that PQLC2 produced in yeast behaves like the endogenous Ypq[3]: it is sorted to the vacuole via the ALP pathway in a manner dependent on proper recognition of its dileucine motif by the AP-3 adaptor complex
Previous work in yeast has highlighted the role of the AP-3 complex in the vacuolar targeting of several transmembrane proteins, including alkaline phosphatase (Pho8/ALP)[20,21,22], the Vma[3] SNARE required for vacuole fusion[16,20], casein kinase 3 (Yck3)[23], two proteins acting as putative adaptors for protein ubiquitylation (Sna[2] and − 4)[24,25], the Ncr[1] protein involved in sphingolipid metabolism[26], and a protein involved in autophagy (Atg27)[27]
Summary
These transporters is a triple helix bundle (THB)[10]. In 7-TM transporters such as cystinosin, PQLC2, and SWEET proteins, a linker TM (TM4) connects two THBs (Fig. 1C), whereas the 3-TM semiSWEET monomers associate into 6-TM dimers where the interface between the monomers provides the transport conduit[8,10]. The ypq2∆ mutant is resistant to the toxic arginine analogue canavanine, a phenotype compatible with a role of Ypq[2] in arginine (and canavanine) export from the vacuole This phenotype is complemented by rat PQLC2, shown in electrophysiology and uptake experiments to catalyze export of basic amino acids and canavanine. This suggests that yeast Ypq[2] and PQLC2 are functional orthologs[6]. Our results show that the Ypq proteins preferentially use the pathway depending on the AP-3 adaptor complex and on an acidic dileucine motif present in their second cytosolic loop. The results of further experiments in HeLa cells reveal that the AP-3 complex is essential to proper localization of PQLC2 to the lysosomal membrane
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