Understanding the mechanism of lipid translocation

P4-ATPases belong to a family of cation-transporting proteins, the P-type ATPases, that are involved in relevant physiological functions, such as the generation of electrochemical gradients across membranes or cell detoxification. P4-ATPases present high sequence similarity to other members of the P-type family. However, they also show remarkable differences, especially with respect to the amino acid residues involved in cation binding.

 

Although this may suggest an adaptation to accomplish lipid translocation, it is not known how these proteins are able to move such large molecules as the phospholipids from one side of the membrane to the other. Notably, a striking feature of P4-ATPases across species is that they form complexes with members of the CDC50 family of small membrane proteins.

 

What is the primary role of the CDC50 protein in the transporter complex? Are P4-ATPases alone sufficient to flip phospholipids or do they rely on CDC50 binding partners for coupling ATP hydrolysis to phospholipid translocation? Are CDC50 proteins involved in lipid binding? Are there other proteins required for lipid translocation? What are the substrate-binding sites within the P4-ATPase sequence?

 

In order to answer these questions, we use directed-mutagenesis studies followed by functional complementation of yeast mutant strains. Further biochemical analysis is accomplished in collaboration with the Biomembranes group at the Transport Biology section.

 

 

 

 

Figure 1: Schematic representation of a P4-ATPase and its Cdc50 interacting-partner. As all other P-type ATPases, P4-ATPases present a series of conserved protein domains: M, transmembrane region; P, Phosphorylation domain, containing an aspartic acid residue that gets phosphorylated during the catalytic cycle; N, nucleotide-binding domain; A, actuator domain involved in dephosphorylation of the P-domain during the catalytic cycle. The arrow shows the direction of lipid translocation