Structure and function analysis of the aminophospholipid translocase TAT-1.

The aminophospholipid translocase TAT-1 maintains phosphatidylserine (PS) asymmetry in the plasma membrane and regulates endocytic transport. Despite these important functions, the structure-function relationship of this protein is poorly understood. Taking advantage of the mutations identified by the million mutation project, we investigated the effects of 16 single amino acid substitutions on the two functions of the TAT-1 protein. Two substitutions that alter a highly conserved PISL motif in the fourth transmembrane domain and a highly conserved DKTGT phosphorylation motif, respectively, disrupt both functions of TAT-1, leading to a vesicular gut defect and ectopic PS exposure on the cell surface, whereas most other substitutions across the TAT-1 protein, often predicted to be deleterious by bioinformatics programs, do not affect the functions of TAT-1. These results provide evidence for the importance of the PISL and DKTGT motifs in P4-type ATPases and improve our understanding of the structure-function relationship of TAT-1. Our study also provides an example of how the million mutation project helps decipher the structure, functions, and mechanisms of action of important genes.