Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross-linking. After cross-linking and immunoblot analysis alkyl-GrnP synthase could be detected in a 210-kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two-dimensional SDS/PAGE demonstrated that GrnP-acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can form a homotrimeric complex in the absence of GrnP-acyltransferase as was demonstrated by immunoblot analysis after cross-linking experiments with either GrnP-acyltransferase deficient human fibroblast homogenates or recombinant (His) 6 -tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthase interacts selectively with GrnP-acyltransferase in a heterotrimeric complex and in the absence of GrnP-acyltransferase can also form a homotrimeric complex.

The structures of the polymorphic forms of clathrin light chains were analyzed by two peptide mapping procedures. Comparison of the products of partial digestion by V8 protease showed no common peptides between LCA and LCB from bovine brain. No similarities between clathrin light chains and tropomyosin chains from bovine brain and skeletal muscle were detected with this technique. The peptides produced by complete tryptic digestion of LCA and LCB from bovine brain and bovine adrenal gland were analyzed by reverse phase h.p.l.c. For both LCA and LCB the polypeptides from different tissues showed considerable homology. LCA from brain and adrenal gland shared 10 out of a total of 15 peptides. LCB from brain and adrenal gland shared 10 out of 14 peptides. In contrast, when LCA was compared with the LCB chain from the same tissue very few peptides were shared; 4/23 for brain and 3/21 for adrenal gland. These results strongly indicate that, within a tissue, LCB is not related to LCA by post‐translational processing and that each chain is encoded by a separate gene. The data also demonstrate the close homology of the different forms of LCA and LCB expressed in different tissues within the same organism. Thus the polymorphic differences of clathrin light chains within a tissue are greater than those between tissues.

Alkyldihydroxyacetonephosphate synthase (alkylglycerone-phosphate synthasej is a peroxisomal enzyme involved in ether phospholipid biosynthesis. The recent cloning of the cDNA encoding this enzyme from guinea pig liver enabled the raising of specific antisera against this enzyme. Both a synthetic peptide corresponding to a predicted epitope and a recombinant protein expressed in Escherichia coli were used for that purpose. Using western blot techniques, the solubilization of the enzyme from the peroxisomal membrane by Triton X-100 in the presence of salt was confirmed. Neutral hydroxylamine treatment of peroxisomes resulted in almost no release of the protein from the membrane. The complete polypeptide chain of the enzyme was resistant to proteolysis by trypsin when intact peroxisomes were studied. Carbonate treatment released alkyldihydroxyacetonephosphate synthase from the membrane indicating that the enzyme is not an integral membrane protein. This idea is in accord with the absence of a clear hydrophobic transmembrane domain in the deduced amino acid sequence of the enzyme. Alkyldihydroxyacetonephosphate synthase, as well as its mRNA, could be detected in all five guinea pig tissues examined. When using the antiserum against guinea pig recombinant alkyldihydroxyacetonephosphate synthase, a cross-reactive protein was detected in a human liver homogenate that runs at a slightly higher molecular mass. The absence of this band in liver of Zellweger syndrome and Rhizomelic chondrodysplasia punctata patients provides strong evidence that it represents the human homolog of this enzyme.Keywords: glycerone-phosphate synthase; ether phospholipid ; peroxisome; membrane association ; peroxisomal disorder, Ether phospholipids comprise a special class of natural phospholipids. In mammals, these have either an alkyl or an alkenyl ether linkage at the sn-I position and an acyl ester linkage at the sn-2 position of the glycerol backbone. Although they represent about 15% of total human phospholipids, until now little has been known about the specific functions of ether phospholipids.A notable exception to this is platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a bioactive phospholipid for which biological roles through specific receptor activations have convincingly been established [I].The first steps of ether phospholipid biosynthesis take place in peroxisomes [2, 31 or the closely related microbody glycosomes [4]. The process starts with the acylation of dihydroxyacetonephosphate (glycerone-Pj by the enzyme dihydroxyacetonephosphate acyltransferase. The ether linkage is then introduced by a second enzyme, alkyldihydroxyacetonephosphate synthase, that catalyzes the exchange of the acyl chain in acyldihydroxyacetone for a long-chain fatty alcohol.The deficiency of ether phospholipids in a number of human peroxisomal disorders has clearly emphasized the indispensible role of peroxisomes for ether lipid synthesis [5]. The prototype Correspondence to H . van

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