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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, allows the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2 (Coleman and Molday, 2011).

Cdc50 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).

References associated with 8.A.27 family:

Coleman, J.A. and R.S. Molday. (2011). Critical role of the β-subunit CDC50A in the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2. J. Biol. Chem. 286: 17205-17216. 21454556
Engel, M., P. Snikeris, N. Matosin, K.A. Newell, X.F. Huang, and E. Frank. (2016). mGluR2/3 agonist LY379268 rescues NMDA and GABAA receptor level deficits induced in a two-hit mouse model of schizophrenia. Psychopharmacology (Berl) 233: 1349-1359. 26861891
Garcia-Sanchez S., Sanchez-Canete MP., Gamarro F. and Castanys S. (2014). Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit. Biochem J. 459(1):83-94. 24447089
Hanson, P.K., L. Malone, J.L. Birchmore, and J.W. Nichols. (2003). Lem3p is essential for the uptake and potency of alkylphosphocholine drugs, edelfosine and miltefosine. J. Biol. Chem. 278: 36041-36050. 12842877
Lenoir G., Williamson P., Puts CF. and Holthuis JC. (2009). Cdc50p plays a vital role in the ATPase reaction cycle of the putative aminophospholipid transporter Drs2p. J Biol Chem. 284(27):17956-67. 19411703
Liu, L., L. Zhang, L. Zhang, F. Yang, X. Zhu, Z. Lu, Y. Yang, H. Lu, L. Feng, Z. Wang, H. Chen, S. Yan, L. Wang, Z. Ju, H. Jin, and X. Zhu. (2017). Hepatic Tmem30a Deficiency Causes Intrahepatic Cholestasis by Impairing Expression and Localization of Bile Salt Transporters. Am J Pathol 187: 2775-2787. 28919113
Misu, K., K. Fujimura-Kamada, T. Ueda, A. Nakano, H. Katoh, and K. Tanaka. (2003). Cdc50p, a conserved endosomal membrane protein, controls polarized growth in Saccharomyces cerevisiae. Mol. Biol. Cell 14: 730-747. 12589066
Perandrés-López, R., M.P. Sánchez-Cañete, F. Gamarro, and S. Castanys. (2018). Functional role of highly-conserved residues of the N-terminal tail and first transmembrane segment of a P4-ATPase. Biochem. J. [Epub: Ahead of Print] 29438067
Peréz-Victoria, F.J., Sanchez-Canete, M.P., Castanys, S., and Gamarro, F. (2006). Phospholipid translocation and miltefosine potency require both L. donovani miltefosine transporter and the new protein LdRos3 in Leishmania parasites. J. Biol. Chem. 281: 23766-23775. 16785229
Poulsen, L.R., R.L. López-Marqués, S.C. McDowell, J. Okkeri, D. Licht, A. Schulz, T. Pomorski, J.F. Harper, and M.G. Palmgren. (2008). The Arabidopsis P4-ATPase ALA3 Localizes to the Golgi and Requires a β-Subunit to Function in Lipid Translocation and Secretory Vesicle Formation. Plant Cell 20: 658-676. 18344284
Riekhof, W.R. and Voelker, D.R. (2006). Uptake and utilization of lyso-phosphatidylethanolamine by Saccharomyces cerevisiae. J. Biol. Chem. 281: 36588-36596. 17015438
Segawa, K., S. Kurata, Y. Yanagihashi, T.R. Brummelkamp, F. Matsuda, and S. Nagata. (2014). Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure. Science 344: 1164-1168. 24904167
Tokai, M., H. Kawasaki, Y. Kikuchi, and K. Ouchi. (2000). Cloning and characterization of the CSF1 gene of Saccharomyces cerevisiae, which is required for nutrient uptake at low temperature. J. Bacteriol. 182: 2865-2868. 10781556
van der Mark, V.A., D.R. de Waart, K.S. Ho-Mok, M.M. Tabbers, H.W. Voogt, R.P. Oude Elferink, A.S. Knisely, and C.C. Paulusma. (2014). The lipid flippase heterodimer ATP8B1-CDC50A is essential for surface expression of the apical sodium-dependent bile acid transporter (SLC10A2/ASBT) in intestinal Caco-2 cells. Biochim. Biophys. Acta. 1842: 2378-2386. 25239307
Wang, J., Q. Wang, D. Lu, F. Zhou, D. Wang, R. Feng, K. Wang, R. Molday, J. Xie, and T. Wen. (2017). A biosystems approach to identify the molecular signaling mechanisms of TMEM30A during tumor migration. PLoS One 12: e0179900. 28640862