Alkyl-dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes

Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl-GrnP synthase a re the key enzymes involved in the biosynthesis of ether phospholipids. Bot h enzymes are located on the inside of the peroxisomal membrane. Here we re port evidence for a direct interaction between these enzymes obtained by th e use of chemical cross-linking. After cross-linking and immunoblot analysi s alkyl-GrnP synthase could be detected in a 210-kDa complex which was loca ted entirely on the lumenal side of the peroxisomal membrane. Two-dimension al SDS/PAGE demonstrated that GrnP-acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes in teract, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can f orm a homotrimeric complex in the absence of GrnP-acyltransferase as was de monstrated by immunoblot analysis after cross-linking experiments with eith er GrnP-acyltransferase deficient human fibroblast homogenates or recombina nt (His)(6)-tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthas e interacts selectively with GrnP-acyltransferase in a heterotrimeric compl ex and in the absence of GrnP-acyltransferase can also form a homotrimeric complex.