8.A.27  The CDC50 P-type ATPase Lipid Flippase β-Subunit (CDC50) Family 

The first characterized member of the phospholipid importer β-subunit of phospholipid-translocating P-type ATPases is the Lem3 (ligand-effect modulator 3) (YNL323W) protein of Saccharomyces cerevisiae defined as the PLI-β family (Hanson et al., 2003). This protein was reported to be responsible for the import of alkylphosphocholine drugs such as edelfosine and miltefosine which have been used in the treatment of protozoal and fungal diseases, particularly leishmaniasis. Mutational loss of Lem3 results in poor uptake of these drugs as well as of fluorescent, short chain, 7-nitrobenz-2-oxo-1,3-diazol-4-yl (NBD)-labeled phosphatidylcholine and NBD-phosphatidylethanolamine. Phosphatidylserine transport appeared to be normal in a lem3 mutant. Lem3 is the prototype for a large family of eukaryotic proteins found in animals, plants, fungi, slime molds, ciliates and protozoans but not in prokaryotes. Lem3 (414 aas) has 2 putative TMSs at residues 74-95 and 373-394 and is homologous to the putative S. cerevisiae cell division protein, Cdc50 (391 aas; P25656) and an uncharacterized paralogue, Ynr048w of 393 aas; P53740. Lem3 serves as the β-subunit for both Dnf1 (3.A.3.8.4) and Dnf2 (3.A.3.8.5), two phospholipid flipping P-type ATPases in S. cerevisiae (Riekhof and Voelker, 2006). These proteins may generally be β-subunits of phospholipid-translocating P-type ATPases (Lenoir et al., 2009). The beta-subunit, CDC50A or TMEM30a, allows the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2 (Coleman and Molday, 2011).  Loss of Tmem30a in human RBCs results in dendritic sprouting of rod bipolar cells, increased astrogliosis and RBC death. Thus, Tmem30a plays an essental role in retinal bipolar cells (Yang et al. 2019). TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma (Ennishi et al. 2020).

Cdc50 or TMEM30 is a family of conserved eukaryotic proteins that interact with P4-ATPases (phospholipid translocases). Cdc50 association is essential for endoplasmic reticular export of P4-ATPases and proper translocase activity. García-Sánchez et al. 2014 analysed the role of Leishmania infantum LiRos3, the Cdc50 subunit of the P4-ATPase miltefosine transporter (LiMT), on trafficking and complex functionality using site-directed mutagenesis and domain substitution. They identified 22 invariant residues in the Cdc50 proteins from L. infantum, human and yeast. Seven of these residues are found in the extracellular domain of LiRos3, the conservation of which is critical for ensuring that LiMT arrives at the plasma membrane. The substitution of other invariant residues affected complex trafficking to a lesser extent. Invariant residues located in the N-terminal cytosolic domain play a role in transport activity. Partial N-glycosylation of LiRos3 reduced miltefosine transport, and total N-deglycosylation completely inhibited LiMT trafficking to the plasma membrane. One of the N-glycosylation residues proved to be invariant amoung members of the Cdc50 family. The transmembrane and exoplasmic domains are not interchangeable with the other two L. infantum Cdc50 proteins to maintain LiMT interaction. These findings indicate that both invariant and N-glycosylated residues of LiRos3 are involved in LiMT trafficking and transport activity (βGarcía-Sánchez et al. 2014).