Mapping functional interactions in a heterodimeric phospholipid pump.
Type 4 P-type ATPases (P(4)-ATPases) catalyze phospholipid transport to generate phospholipid asymmetry across membranes of late secretory and endocytic compartments, but their kinship to cation-transporting P-type transporters raised doubts about whether P(4)-ATPases alone are sufficient to mediate flippase activity. P(4)-ATPases form heteromeric complexes with Cdc50 proteins. Studies of the enzymatic properties of purified P(4)-ATPase·Cdc50 complexes showed that catalytic activity depends on direct and specific interactions between Cdc50 subunit and transporter, whereas in vivo interaction assays suggested that the binding affinity for each other fluctuates during the transport reaction cycle. The structural determinants that govern this dynamic association remain to be established. Using domain swapping, site-directed, and random mutagenesis approaches, we here show that residues throughout the subunit contribute to forming the heterodimer. Moreover, we find that a precise conformation of the large ectodomain of Cdc50 proteins is crucial for the specificity and functionality to transporter/subunit interactions. We also identified two highly conserved disulfide bridges in the Cdc50 ectodomain. Functional analysis of cysteine mutants that disrupt these disulfide bridges revealed an inverse relationship between subunit binding and P(4)-ATPase-catalyzed phospholipid transport. Collectively, our data indicate that a dynamic association between subunit and transporter is crucial for the transport reaction cycle of the heterodimer.
Department of Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, The Netherlands.
Catheleyne F
CF
Puts
Radhakrishnan
R
Panatala
Hanka
H
Hennrich
Alina
A
Tsareva
Patrick
P
Williamson
Joost C M
JC
Holthuis
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active / Q000502:physiology; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010449:Peptide Mapping / Q000379:methods; D049294:Phospholipid Transfer Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
C427246:CDC50 protein, S cerevisiae; D046912:Multiprotein Complexes; D049294:Phospholipid Transfer Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases