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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.
Ether phospholipids are a special class of phospholipids which can be divided in two groups. The first group has a saturated ether linkage at the sn-1-position of the glycerol moiety, whereas the second group, also known by their trivial name plasmalogens, has an α,β-unsaturated ether linkage at that position. Although ether phospholipids make up a considerable portion of the phospholipid mass in mammals little is known about their physiological function . Proposed functions include a role in signal transduction , prostaglandin production and arachidonic acid metabolism , protection against reactive oxygen species  and protein secretion .
The biosynthesis of ether phospholipids in higher organisms starts with the acylation of dihydroxyacetonephosphate (GrnP) by GrnP-acyltransferase to form acyl-GrnP[6,7]. The acyl moiety is then replaced by a long chain fatty alcohol yielding alkyl-GrnP. This reaction, in which the ether linkage is introduced, is catalysed by alkyl-GrnP synthase [6,7]. Biochemical as well as immunocytochemical experiments have shown that these enzymes appear to be membrane-bound and are localized entirely on the lumenal side of the peroxisomal membrane [8–11].
The important role of peroxisomes in ether phospholipid biosynthesis in humans is emphasized by the deficiency of ether phospholipids in a group of genetic diseases called peroxisomal disorders . These disorders can be subdivided into three groups depending on whether there is a generalized (group A), multiple (group B) or single loss (group C) of peroxisomal functions . The Zellweger syndrome, the most well known representative of all peroxisomal disorders, is characterized by an accumulation of very long chain fatty acids, pristanic acids, phytanic acids and bile acid intermediates and by the absence of ether phospholipids and morphologically detectable peroxisomes [14–16].
In Rhizomelic chondrodysplasia punctata (RCDP), an example of group B peroxisomal disorders, peroxisomes are morphologically intact but five biochemical abnormalities are found including a deficiency of GrnP-acyltransferase, alkyl-GrnP synthase, phytanoyl-CoA hydroxylase and mevalonate kinase and the absence of the mature form of peroxisomal 3-ketoacyl-CoA thiolase [17,18]. Because the genetic defect in RCDP is a mutation in the receptor for the peroxisomal targeting signal (PTS)2 [19–21], it is likely that the enzymatic deficiencies found are due to the inability of peroxisomes to import PTS2-containing proteins. The molecular cloning of the cDNAs of alkyl-GrnP synthase [22,23], phytanoyl-CoA hydroxylase [24,25] and peroxisomal 3-ketoacyl-CoA thiolase  indeed revealed the presence of a cleavable presequence, containing the PTS2 signal, and explained their deficiency in RCDP.
The cDNA of GrnP-acyltransferase has also recently been cloned [9,27]. Surprisingly the nucleotide-derived amino acid sequence revealed a protein containing a PTS1 signal. The deficiency of GrnP-acyltransferase in RCDP strongly suggests that the enzyme is somehow dependent on the PTS2 import pathway, despite the absence of a PTS2 signal in the protein itself. Support for this relationship was provided recently when it was demonstrated that GrnP-acyltransferase activity is dependent on the presence of the alkyl-GrnP synthase protein, being either active or inactive (E. C. J. M. de Vet, L. Ijlst, W. Oostheim, C. Dekker, H. W. Moser, H. van den Bosch & R. J. A. Wanders, unpublished data). These results, suggesting a possible interaction of the enzymes, were confirmed biochemically when the enzymes were isolated from extracts of rabbit Harderian gland peroxisomes as a trimeric complex . A close interaction of the enzymes (allowing substrate channelling) was also suggested by kinetic experiments, which indicated that endogenously generated acyl-GrnP was used in preference to exogenously added substrate .
In this study the possible protein–protein interactions of alkyl-GrnP synthase have been investigated by using biochemical experiments such as chemical cross-linking and coimmunoprecipitation. Evidence is provided that alkyl-GrnP synthase can form both a homotrimeric as well as a heterotrimeric complex.
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- Materials and methods
In this study we have demonstrated that a distinct alkyl-GrnP synthase immunoreactive complex of 210 kDa was recovered following chemical cross-linking. This complex was observed clearly only with the reagents BMH and DPDPB (Fig. 1), indicating that the chemical groups with which the cross-linker reacts and the length of the cross-linker arm influence alkyl-GrnP synthase cross-linking to interacting proteins. Using cross-linking and immunoblot analysis, the complex was not only detected in a guinea-pig liver mitochondrial fraction, but also in this fraction from several other guinea-pig tissues and in the supernatant of detergent-solubilized peroxisomes. This shows that the complex does not easily dissociate in solubilized preparations. In addition, complexes of similar size were detected in human fibroblasts (Fig. 4).
Alkyl-GrnP synthase activity measurements after cross-linking showed that treatment with amine-reactive cross-linkers hardly affected activity but treatment with sulphydryl-reactive reagents abolished activity completely. This result is in agreement with previous observations that alkyl-GrnP synthase is sensitive to sulphydryl reagents  and that enzyme activity is stimulated by dithiothreitol . In a recent report it was shown that saturating amounts of the substrate palmitoyl-GrnP protected alkyl-GrnP synthase against inactivation by N-ethylmaleimide, suggesting that a sulphydryl group necessary for activity of the enzyme is located in the active centre .
By proteolysis experiments (Fig. 2) it was demonstrated that the immunoreactive bands of the complexes did not shift to lower molecular masses when intact peroxisomes were used, indicating that no subunit of the complex has a cytoplasmically exposed domain.
Several investigations have suggested that alkyl-GrnP synthase may form a heteromultimeric complex with GrnP-acyltransferase. Hardeman et al.  showed that endogenously generated substrate was used at a higher efficiency when compared to exogenously added substrate, suggesting a close interaction between the two enzymes.
Thai et al.  obtained further evidence for such interactions during purification of GrnP-acyltransferase from rabbit Harderian gland peroxisomes. After detergent solubilization the enzyme was isolated as a trimeric complex of about 210 kDa by sucrose density gradient centrifugation. Upon further purification, this complex was found to consist of two proteins, namely alkyl-GrnP synthase and GrnP-acyltransferase.
To collect additional information about the complex formation of both enzymes from sources other than Harderian glands two independent experimental approaches were used: chemical cross-linking and coimmunoprecipitation. On immunoblots of peroxisome-enriched fractions from guinea-pig liver after cross-linking, the monomeric immunoreactive band of GrnP-acyltransferase had disappeared, but no (hetero)multimeric complexes with this enzyme could be detected. The possibility that this resulted from shielding of the epitope recognized by the antibodies, due to conformational changes after cross-linking, was investigated by using two-dimensional SDS/PAGE. Samples were cross-linked with a reducible cross-linker, electrophoresed in the first dimension, reduced and subsequently electrophoresed in the second dimension. Both enzymes (Fig. 3) were detected on immunoblots and were present in a high molecular weight complex in the range of 210 kDa before reduction, indicating that they might form a heterotrimeric complex of this size.
Chemical cross-linking was also performed in human fibroblasts, either from controls having GrnP-acyltransferase or from patients lacking this enzyme. A complex of about 210 kDa was detected in the cell lines containing the GrnP-acyltransferase protein (Fig. 4). Surprisingly, in the cell lines missing the GrnP-acyltransferase protein, we also detected a complex of about 210 kDa, but clearly running at a slightly lower position. It is possible that in the absence of GrnP-acyltransferase another protein of an almost identical size fills up the place, giving rise to the formation of yet another complex. This possibility was confirmed by our observation that recombinant (His)6-tagged alkyl-GrnP synthase forms homodimers and homotrimers after cross-linking in an E. coli supernatant as well as after immobilization on Ni2+-ProBond resin beads (Fig. 5). After incubation of those beads with a Chaps-extract of peroxisomes subsequent cross-linking caused the formation of an additional immunoreactive band that probably corresponds to the heterotrimeric complex containing GrnP-acyltransferase from the detergent extract bound to the immobilized alkyl-GrnP synthase.
Finally, in coimmunoprecipitation experiments (Fig. 6) using Chaps-extracts of peroxisomes, it was found that at least half of the alkyl-GrnP synthase pool was removed with GrnP-acyltransferase immunoprecipitates and vice versa. Complete coimmunoprecipitation may have been prevented by dissociation of the complex in the detergent extract or to differential solubilization of the two enzymes in the detergent extract.
Taken together, this report provides several lines of evidence that alkyl-GrnP synthase and GrnP-acyltransferase, peroxisomal enzymes involved in ether phospholipid biosynthesis, form a heterotrimeric complex. At low BMH concentrations an intermediate dimeric complex was detected also (Fig. 1). This complex formation does not appear to be necessary for the activity of both enzymes. This was shown recently by radiation inactivation experiments, which demonstrated that the active functional units of both enzymes were the monomers . However, complex formation between the two enzymes is likely to facilitate substrate channelling, as is frequently observed in nature.