Cdc50p plays a vital role in the ATPase reaction cycle of the putative aminophospholipid transporter Drs2p.
Members of the P(4) subfamily of P-type ATPases are believed to catalyze transport of phospholipids across cellular bilayers. However, most P-type ATPases pump small cations or metal ions, and atomic structures revealed a transport mechanism that is conserved throughout the family. Hence, a challenging problem is to understand how this mechanism is adapted in P(4)-ATPases to flip phospholipids. P(4)-ATPases form heteromeric complexes with Cdc50 proteins. The primary role of these additional polypeptides is unknown. Here, we show that the affinity of yeast P(4)-ATPase Drs2p for its Cdc50-binding partner fluctuates during the transport cycle, with the strongest interaction occurring at a point where the enzyme is loaded with phospholipid ligand. We also find that specific interactions with Cdc50p are required to render the ATPase competent for phosphorylation at the catalytically important aspartate residue. Our data indicate that Cdc50 proteins are integral components of the P(4)-ATPase transport machinery. Thus, acquisition of these subunits may have been a crucial step in the evolution of flippases from a family of cation pumps.
Lenoir
Guillaume
G
Department of Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
Williamson
Patrick
P
Puts
Catheleyne F
CF
Holthuis
Joost C M
JC
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain / Q000502:physiology; D066298:In Vitro Techniques; D046912:Multiprotein Complexes / Q000378:metabolism; D016296:Mutagenesis; D010766:Phosphorylation / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
C427246:CDC50 protein, S cerevisiae; C104014:DRS2 protein, S cerevisiae; D046912:Multiprotein Complexes; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases