Disease mutation study identifies essential residues for phosphatidylserine flippase ATP11A

PS flippase (P4-ATPase) transports PS from the outer to the inner leaflet of the lipid bilayer in the membrane to maintain PS asymmetry, which is important for biological activity of the cell. ATP11A is expressed in multiple tissues and plays a role in myotube formation. However, detailed cellular function of ATP11A remains elusive. Mutation analysis revealed that I91, L308 and E897 residues in ATP8A2 are important for flippase activity. In order to investigate the roles of these corresponding amino acid residues in ATP11A, we assessed the expression and flippase activity of the respective ATP11A mutant proteins. ATP11A mainly localizes to the Golgi when co-expressed with TMEM30A, the β-subunit of the complex. Y300F and D913K mutations affect correct Golgi localization and PS stimulated flippase activity. In addition, Y300F mutation causes reduced ATP11A expression. Our data provides insight into essential residues for expression and flippase activity of ATP11A.

Thus far, the physiological functions of the majority of mammalian P4-ATPases were still unclear. Only mutations of several P4-ATPases were reported to cause severe human disease [30]. For instance, mutations identified in ATP8B1 are associated with liver disorders such as progressive familial intrahepatic cholestasis and hearing loss [31,32]. Mutations in ATP8A2 cause severe neurological disorders such as cerebellar ataxia, mental retardation and disequilibrium syndrome [33][34][35][36]. ATP11C play a crucial role in differentiation of B cell in lymphopenia, anemia and intrahepatic cholestasis in mice 4 [37][38][39][40]. ATP11A exhibits largely similar cellular distribution with ATP8A1 and ATP8A2 [22,41]. However, only a few studies were reported on ATP11A. ATP11A is ubiquitously expressed in various tissues [22] and deletion of Atp11a results in lethality during embryogenesis. Recent research indicated that the phospholipid flippase complex of ATP11A and CDC50A acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation [42]. The detailed cellular function of ATP11A remains to be determined.
Previous study have reported ATP8B1 mutations L127P and E981K in patients with familial intrahepatic cholestasis type 1 (PFIC1), while the mutation I344F is identified in patients with benign recurrent intrahepatic cholestasis type 1 (BRIC1) [43,44]. An ATP8B1 homozygous mutation L127V causes intrahepatic cholestasis in two Omani siblings [45]. The equivalent mutations of bovine ATP8A2 are I91P, I91V, L308F and E897K. To elucidate the functional consequences of flippase disease mutations, Rasmus H. et al. investigated the effect of mutations of those residues on expression and activity of ATP8A2 and found out the essential roles of these residues to the flippase translocation process [46]. Mutations of the I91 and L308 residues in ATP8A2 are positioned near proposed translocation routes in the protein. Mutation of the E897 residue is located at the exoplasmic loop between transmembrane helix 5 and 6. This mutational analysis suggested that I91, L308 and E897 residues affected the transport of phospholipids.
In order to investigate the effect of these above mentioned corresponding amino acid residues of ATP11A in the transporting phospholipids process, we matched these mutations to the equivalent sites of human ATP11A, which are I80P, I80V, Y300F and D913K respectively, and introduced those mutations into ATP11A ORF by site-direct mutagenesis technique. By investigating the expression pattern and flippase activity of these mutated ATP11A proteins, we demonstrated that variants of Y300F and D913K affected correct Golgi localization and the amount of PS internalization in plasma membrane. Additionally, Y300F mutation led to a decrease in ATP11A expression.
This data provides insight into residues important for expression and flippase activity of ATP11A protein.

Site-directed mutagenesis
Primers carrying the mutations were designed using NEBaseChanger software. Mean fluorescence intensity was calculated for each group.

Statistical analysis
Statistical analysis was performed by one-way analysis of variance or student's t-test using GraphPad Prism 6 software. The differences were considered statistically significant at P values <0.05. The quantitative data are presented as the mean ±SEM as indicated in the figure legends. All experiments were performed in triplicate and repeated at least twice.

Conservative analysis of ATP11A mutational sites
Previous studies [43,44] reported that the ATP8B1 mutational sites found in patients, L127, I344 and E897, are located in exons 4, 12, and 24, respectively. And these mutations are highly conserved among 10 different species (Fig. 1A, B).
Comparative amino acid sequence alignment of other ATP11A proteins across different species revealed that the I80, Y300 and D913 mutations occurred also at highly conserved positions (Fig. 1A), which located in exons 3, 11, and 24, respectively (Fig.   1B). Topology of ATP11A has ten transmembrane helices and A, P and N domains and the C-terminal regulatory domain [47,48] (Fig. 1C). Mutation sites of I80 and Y300 are located in the transmembrane region, while mutation sites of D913 is located at the exoplasmic loop between transmembrane helices 5 and 6 (Fig. 1C). These data demonstrated that the corresponding mutational sites in ATP11A are crucial for the conformational stability of its transmembrane structure.

The Y300F variant of ATP11A impairs its expression level
To investigate the expression levels of ATP11A variants, we transfected plasmids  2). Considering the membrane proteins generally undergo glycosylatation process before maturation [25,49], the detected 145kDa ATP11A should be the mature glycosylated isoform. The immunoblotting data manifested that the expression level of ATP11A-Y300F in HEK 293T cells was reduced ~40% compared to ATP11A-WT ( Fig. 2), while that of other ATP11A variants seems unchanged. The reduced content of ATP11A-Y300F likely resulted from the proteasomal protein degradation, which triggered by protein misfolding [50,51].

ATP11A in the presence of TMEM30A
In order to detect the effect of mutations on the cellular localization of ATP11A, we first investigated the intracellular localization of variants by immunocytochemistry in the context of transfection of ATP11A without TMEM30A in COS7 cells.
ATP11A-WT was largely localized to the endoplasmic reticulum (ER), which was manifested by the specific ER marker calnexin, and its mutants presented similar localization pattern (Fig. S1), suggesting little impact of these variants in ATP11A subcellular localization without the help of TMEM30A.
Previous studies have indicated that TMEM30A play a key role in the correct positioning of flippases [52,53]. To this end, we further investigated the effect of TMEM30A co-expression on the subcellular localization of ATP11A by confocal microscopy. As mentioned above, when ATP11A and its mutants were single-transfected into COS7 cells, they were largely localized to ER (Fig. S1).
However, in the presence of TMEM30A, large extent of ATP11A exit from ER to Golgi. This indicated that TMEM30A might help the correct positioning of ATP11A.
We further investigated the impact of variants on positioning with the help of TMEM30A. Notably, ATP11A-I80P and ATP11A-I80V mostly restricted to Golgi apparatus, while ATP11A-Y300F and ATP11A-D913K still resided at ER improperly ( Fig. 3A). Statistical data manifested that the colocalization rate of ATP11A-WT, ATP11A-I80P and ATP11A-I80V with Golgi was about 60-70%, while that of ATP11A-Y300F and ATP11A-D913K with Golgi was only ~20-30% (Fig. 3B). These  4F). These data demonstrated that variants of Y300F and D913K resulted in the reduced PS internalization in plasma membrane, which might be caused by a reduction in the amount of ATP11A or a decrease in the activity of ATP11A on the plasma membrane.

Discussion
Eukaryotic P4-ATPases plays important roles in various cellular processes. Atp8a2 was reported to play a role in promoting neurite outgrowth in neuronal PC12 cells and rat hippocampal neurons [54]. Studies in mouse models have contributed to our understanding of the physiological functions of mammalian P4ATPases: Atp8a1-deficient mice exhibit delayed hippocampus dependent learning, Atp8a2-mutant mice display neurological abnormalities and Atp11c-deficient mice show arrested B-cell development [30]. However, little is known about the in vivo function of ATP11A. In order to get a deeper understanding of ATP11A, we selected three mutation sites L127, I344, E981 screened by ATP8B1, a member of the homologous family, as a reference.
An ATP8B1 homozygous mutation ATP8B1-L127V causes intrahepatic cholestasis in two Omani siblings [45]. Mutation of ATP8B1-L127P has been reported to be related to PFIC1, but this mutant did not cause any change in the expression level and subcellular localization of ATP8B1 [43,51]. ATP8B1-I344F detected in European families can cause BRIC1 [43]. A case of PFIC1 with mutation of ATP8B1-E981K in Japanese family was reported [44].
According to the amino acid sequence homology alignment, the equivalent positions of I80P, I80V, Y300F, D913K were determined in ATP11A. Mutant expression plasmids were constructed in vitro and its expression level, localization and activity of flippase in the cell were explored. We found that the expression of variant of Y300F was reduced by 40% by immunoblotting (Fig. 2), indicating that mutation at this site may lead to degradation of ATP11A protein. This mutation site is located in the third transmembrane domain (M3) of ATP11A protein (Fig. 1C) and is highly conserved in all 10 species sequences (Fig. 1A). In the crystal model, phenylalanine cannot be properly linked to isoleucine at position 359 on the M3-M4 loop (Fig. S2F).
This indicates that mutations affecting this residue may lead to protein misfolding and eventually degradation by the proteasome.
In addition, the immunocytochemistry experiments demonstrated that variants of Y300F and D913K could not be correctly located in the Golgi apparatus when co-transfected with TMEM30A (Fig. 3A). Sites of Y300 and D913 are located at M3 and M5-M6 loop of ATP11A protein, respectively (Fig. 1C). In the crystal model, the variant Y300F causes the M3 and M3-M4 loop to be incorrectly connected (Fig. S2F), and variant D913K causes an error in the connection of M6 to M5-M6 loop (Fig. S2H).
M3-M4 and M5-M6 loops are on the cell surface (Fig. 1C). It has been reported that in the extracellular region, the CDC50A ectodomain covers all of the extracellular loops of ATP8A1, except for the M1-2 loop, interacting in an electrostatic complementary manner: the extracellular loops of ATP8A1 bear negative charges, whereas CDC50A bears positive charges [47]. Therefore, we speculated that these two mutations interfere with the normal binding of ATP11A and TMEM30A and eventually cause ATP11A to be incorrectly located. Variants I80P and I80V only altered the connection of adjacent amino acids, but did not change the connection between the M1 and other loops (Fig.   S2C, D). This also explains that these two variants have little effect on the ATP11A 11 protein.
Flippase activity assay revealed that Y300F and D913K mutations resulted in reduced PS internalization in plasma membrane. These data demonstrated that variants of Y300F and D913K may resulted in the reduced PS internalization simply caused by reduced protein amounts in plasma membrane, because these mutant were not properly localized Golgi apparatus. These mutant also may directly reduce activity of the flippase in plasma membrane, eventually leading to a decrease in the amount of PS internalization [55,56].
In summary, our data indicated that Y300F mutation of ATP11A could cause a decrease in ATP11A intracellular content, and variants of Y300F and D913K affected the subcellular localization and flippase activity of ATP11A. This indicates that the Y300 and D913 residues play an important role in the normal function of the ATP11A protein. Besides, we also verified that human TMEM30A protein is essential for the proper localization of ATP11A. These data provide basis for its pathogenic phenotype studies and give insight to its detailed cellular function.