title: C-terminus of the P4-ATPase ATP8A2 functions in protein folding and regulation of phospholipid flippase activity.
abstract: ATP8A2 is a P4-ATPase that flips phosphatidylserine and phosphatidylethanolamine across cell membranes. This generates membrane phospholipid asymmetry, a property important in many cellular processes, including vesicle trafficking. ATP8A2 deficiency causes severe neurodegenerative diseases. We investigated the role of the C-terminus of ATP8A2 in its expression, subcellular localization, interaction with its subunit CDC50A, and function as a phosphatidylserine flippase. C-terminal deletion mutants exhibited a reduced tendency to solubilize in mild detergent and exit the endoplasmic reticulum. The solubilized protein, however, assembled with CDC50A and displayed phosphatidylserine flippase activity. Deletion of the C-terminal 33 residues resulted in reduced phosphatidylserine-dependent ATPase activity, phosphatidylserine flippase activity, and neurite extension in PC12 cells. These reduced activities were reversed with 60- and 80-residue C-terminal deletions. Unlike the yeast P4-ATPase Drs2, ATP8A2 is not regulated by phosphoinositides but undergoes phosphorylation on the serine residue within a CaMKII target motif. We propose a model in which the C-terminus of ATP8A2 consists of an autoinhibitor domain upstream of the C-terminal 33 residues and an anti-autoinhibitor domain at the extreme C-terminus. The latter blocks the inhibitory activity of the autoinhibitor domain. We conclude that the C-terminus plays an important role in the efficient folding and regulation of ATP8A2.
authors.affiliation: Department of Biochemistry and Molecular Biology, Centre for Macular Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Madhavan
authors.initials: M
authors.lastname: Chalat
authors.affiliation: Department of Biochemistry and Molecular Biology, Centre for Macular Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Kody
authors.initials: K
authors.lastname: Moleschi
authors.affiliation: Department of Biochemistry and Molecular Biology, Centre for Macular Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada molday@mail.ubc.ca.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Molday
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology*; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology; D016716:PC12 Cells; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology*; D010743:Phospholipids / Q000378:metabolism; D000072417:Protein Domains; D017510:Protein Folding; D021381:Protein Transport; D051381:Rats
chemical_list: D008565:Membrane Proteins; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; C526978:TMEM30a protein, human; C483858:phosphatidylethanolamine; D000251:Adenosine Triphosphatases; C545445:ATP8A2 protein, human