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).



Chen, B., Y. Jiang, S. Zeng, J. Yan, X. Li, Y. Zhang, W. Zou, and X. Wang. (2010). Endocytic sorting and recycling require membrane phosphatidylserine asymmetry maintained by TAT-1/CHAT-1. PLoS Genet 6: e1001235.

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.

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.

Ennishi, D., S. Healy, A. Bashashati, S. Saberi, C. Hother, A. Mottok, F.C. Chan, L. Chong, L. Abraham, R. Kridel, M. Boyle, B. Meissner, T. Aoki, K. Takata, B.W. Woolcock, E. Viganò, M. Gold, L.L. Molday, R.S. Molday, A. Telenius, M.Y. Li, N. Wretham, N. Dos Santos, M. Wong, N.N. Viller, R.A. Uger, G. Duns, A. Baticados, A. Madero, B.N. Bristow, P. Farinha, G.W. Slack, S. Ben-Neriah, D. Lai, A.W. Zhang, S. Salehi, H.P. Shulha, D.S. Chiu, S. Mostafavi, A.S. Gerrie, D.W. Huang, C. Rushton, D. Villa, L.H. Sehn, K.J. Savage, A.J. Mungall, A.P. Weng, M.B. Bally, R.D. Morin, G.V. Cohen Freue, L.M. Staudt, J.M. Connors, M.A. Marra, S.P. Shah, R.D. Gascoyne, D.W. Scott, and C. Steidl. (2020). TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma. Nat. Med. 26: 577-588.

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.

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.

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.

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.

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.

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]

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.

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.

Riekhof, W.R. and Voelker, D.R. (2006). Uptake and utilization of lyso-phosphatidylethanolamine by Saccharomyces cerevisiae. J. Biol. Chem. 281: 36588-36596.

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.

Timcenko, M., J.A. Lyons, D. Januliene, J.J. Ulstrup, T. Dieudonné, C. Montigny, M.R. Ash, J.L. Karlsen, T. Boesen, W. Kühlbrandt, G. Lenoir, A. Moeller, and P. Nissen. (2019). Structure and autoregulation of a P4-ATPase lipid flippase. Nature 571: 366-370.

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.

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.

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.

Wunderlich, J. (2022). Updated List of Transport Proteins in. Front Cell Infect Microbiol 12: 926541.

Yang, Y., W. Liu, K. Sun, L. Jiang, and X. Zhu. (2019). Tmem30a deficiency leads to retinal rod bipolar cell degeneration. J Neurochem 148: 400-412.


TC#NameOrganismal TypeExample

Alkylphosphocholine resistance/Ro-sensitive-3/Brefeldin-A sensitivity-3 protein, Lem3 or Cdc50 (Hanson et al., 2003).  The 3-D structure of Lem3 complexed with Drs2 (TC# 3.A.3.8.2) has been solved by cryoEM (Timcenko et al. 2019).


Lem3 of Saccharomyces cerevisiae (414 aas; P42838)


The cell division control Cdc50 protein of 391 aas and 3 TMSs. It is an endosomal protein that regulates polarized cell growth (Misu et al. 2003).


Cdc50 of Saccharomyces cerevisiae (P25656)


The miltefosine transporter β-subunit, LdRos3, of phospholipid transporting P-type ATPase-3, LdMT (see TC# 3.A.3.8.19) (Peréz-Victoria et al., 2006; Perandrés-López et al. 2018).


LdRos3 of Leishmania donovani (365 aas; ABB05176)

8.A.27.1.4The plant β-subunit ALIS1 (350aas; two probable TMSs) (functions with the α-subunit of TC#3.A.3.8.6) (Poulsen et al., 2008)PlantsALIS1 of Arabidopsis thaliana (Q9LTW0)

CDC50A or TMEM30A of 361 aas and 2 TMSs.  Required for targetting of the ATP11C ATPase (3.A.3.8.14) and probably several other phospholipid flipping ATPases to the plasma membrane, and possibly also for their activities (Segawa et al. 2014).  Forms a heterodimer with ATP8B1 (van der Mark et al. 2014). Signaling networks are regulated by TMEM30A during cell migration, reflecting the regulatory mechanisms underlying tumor migration (Wang et al. 2017). TMEM30A deficiency leads to intrahepatic cholestasis in mice by impairing the expression and localization of bile salt transporters and the expression of related nuclear receptors (Liu et al. 2017). The 3-D strcutures of 6 distinct intermediates (2.6 - 3.3 Å resolution) of the complex of this protein with ATP8A1 (TC# 3.A.3.8.13) have been solved, revealing the transport cycle for lipid flipping (Hiraizumi et al. 2019). TMEM30A maintains the asymmetric distribution of phosphatidylserine, an 'eat-me' signal recognized by macrophages. TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma (Ennishi et al. 2020).


CDC50A of Homo sapiens


CDC50B or TMEM30B of 351 aas and 2 TMSs.  CDC50 homologues are required for plasma membrane targetting and activity of phospholipid flipping ATPases of subfamily 3.A.3.8 (Segawa et al. 2014).


CDC50B of Homo sapiens


Non-catalytic subunit of the Dnf3 PL-flipping P-type ATPase (TC# 3.A.3.8.20), Crf1 of 393 aas and 2 TMSs at the N- and C-termini.  Also required for nutrient uptake at low temperatures (Tokai et al. 2000).

Crf1 of Saccharomyces cerevisiae


CDC50 family protein, Chat-1 of 348 aas and 2 TMSs, N- and C-terminal.  It is a chaperone protein for the phospholipid-transporting ATPase tat-1 (TC# 3.A.3.8.15) that regulates cell membrane structure and function. It plays a role in maintaining the membrane phosphatidylserine asymmetry and the formation of the tubular membrane structure. It is involved in membrane trafficking and is specifically involved in the recycling and degradation of endocytic cargo with Tat-1 (Chen et al. 2010).

Chat-1 of Caenorhabditis elegans


Zinc transporter, CDF, CDC50, LEM3 or ZIP3, of 471 aas with 2 TMSs at residues 180 and 440 (Wunderlich 2022). 

ZIP3 of Plasmodium falciparum