Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics.

Background : The enzymatic activities of ATP10 family of mammalian P4-ATPases are unknown. Results : ATP10A catalyzes flipping of NBD-PC. Expression of ATP10A altered cell shape, and inhibited cell adhesion and spreading. Conclusions : The enhanced PC-flipping activity by ATP10A changes the lipid composition which may cause a delay in cell spreading. Significance : This is the first evidence showing that PC-flipping activity by P4-ATPase is associated with the plasma membrane dynamics. Abstract We showed previously that ATP11A and ATP11C have flippase activity toward

In this study, we characterized the
ABCB4 cDNA was a kind gift from Kazumitsu Ueda (Kyoto University). The pCAG-based vector for expression of ABCB4 with a Cterminal HA tag was constructed. ABCB4 cDNA was cloned into the pENTR3C vector (Invitrogen), and the pCAG-HA-based vector was prepared as described previously (18).
Transfer of the ABCB4 cDNA to expression vectors was performed using the Gateway system (Invitrogen).    A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp

ATP10A, ATP10B, and ATP10D
Previously, we demonstrated that CDC50 is protein (see Figure 1), stably expressed ATP10A and ATP10D were detected at the plasma membrane in the absence of exogenous CDC50A expression (see Figure 5, A, a, a', b, b', e, and e').
As described previously (19), this probably reflects the fact that the endogenous level of  A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp

Discussion
In this study, we demonstrated that class 5 P4-ATPases (ATP10A, ATP10B, and ATP10D) require their interaction with CDC50A for their exit from the ER and localization to specific cellular compartments where they exert their functions ( Figure 1). Importantly, we revealed that ATP10A has PC-specific flipping activity.
Moreover, the enhanced PC-flipping activity resulting from expression of ATP10A leads to changes in cell shape and delays cell adhesion and spreading.
Unlike ATP8B1 and ATP11A, members of the ATP10 family (especially ATP10B and ATP10D) associate with glycosylated CDC50A with low affinity (Figure 2). Even if the interaction between ATP10 proteins and CDC50A is not as strong as that between ATP8B1 or ATP11A and CDC50A, the interaction is nonetheless critical for the localization of ATP10 proteins to their final destinations: the plasma membrane for ATP10A and ATP10D, and late endosomes for ATP10B (Fig. 1). In support of this result, these P4-ATPases did not exit the ER in cells depleted of CDC50A ( Figure 5A). By      shown. Graphs display averages from three independent experiments ± SD (*p < 0.05, **p < 0.01). Fluor 488-conjugated anti-CD147 antibody prior to permeabilization, and with anti-HA antibody after permeabilization, as described in the legend for Figure 1D and F. Bar, 20 m. (BE) siRNA-treated cells were incubated with the indicated NBD-lipids at 15°C, and the residual fluorescence intensity associated with the cells was determined by flow cytometry as described in the legend for Figure 3.
Fold increase in NBD-lipid uptake compared with vector-infected and siLacZ-treated control cells is shown. Graphs display averages from three independent experiments ± SD (*p < 0.05, **p < 0.01). (F) A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp siRNA-treated cells were lysed to isolate total RNAs, which were processed for RT-PCR.