Phospholipid Flippases Lem3p-Dnf1p and Lem3p-Dnf2p Are Involved in the Sorting of the Tryptophan Permease Tat2p in Yeast

The type 4 P-type ATPases are flippases that generate phospholipid asymmetry in membranes. In budding yeast, heteromeric flippases, including Lem3p-Dnf1p and Lem3p-Dnf2p, translocate phospholipids to the cytoplasmic leaflet of membranes. Here, we report that Lem3p-Dnf1/2p are involved in transport of the tryptophan permease Tat2p to the plasma membrane. The lem3 Δ mutant exhibited a tryptophan requirement due to the mislocalization of Tat2p to intracellular membranes. Tat2p was relocalized to the plasma membrane when trans -Golgi network (TGN)-to-endosome transport was inhibited. Inhibition of ubiquitination by mutations in ubiquitination machinery also rerouted Tat2p to the plasma membrane. Lem3p-Dnf1/2p are localized to endosomal/TGN membranes in addition to the plasma membrane. Endocytosis mutants, in which Lem3p-Dnf1/2p are sequestered to the plasma membrane, also exhibited the ubiquitination-dependent missorting of Tat2p. These results suggest that Tat2p is ubiquitinated at the TGN and missorted to the vacuolar pathway in the lem3 Δ mutant. The NH 2 -terminal cytoplasmic region of Tat2p containing ubiquitination acceptor lysines interacted with liposomes containing acidic phospholipids, including phosphatidylserine. This interaction was abrogated by alanine substitution mutations in the basic amino acids downstream of the ubiquitination sites. Interestingly, a mutant Tat2p containing these substitutions was missorted in a ubiquitination-dependent manner. We propose the following model based on these results; Tat2p is not ubiquitinated when the NH 2 -terminal region is bound to membrane phospholipids, but if it dissociates from the membrane due to a low level of phosphatidylserine caused by perturbation of phospholipid asymmetry in the lem3 Δ mutant, Tat2p is ubiquitinated and then transported from the TGN to the vacuole. Background: Lem3p-Dnf1p and Lem3p-Dnf2p are phospholipid flippases that generate phospholipid asymmetry in yeast. Results: The tryptophan permease Tat2p is missorted from the trans -Golgi network to the vacuole in the lem3 Δ mutant. Conclusion: Phospholipid asymmetry supports plasma membrane transport of Tat2p by inhibiting its improper ubiquitination at the trans -Golgi network. Significance: Phospholipid asymmetry may be involved in proper sorting of membrane proteins.