Proteins and enzymes of the peroxisomal membrane in mammals

Biol Cell (1993) 77, 89-104

89

© Elsevier, Paris

Proteins and enzymes of the peroxisomal membrane in mammals Catherine Causeret, Marc Bentejac, Maurice Bugaut* Laboratoire de Biologie Mol~culaire et Cellulaire, Facult~ des Sciences Mirande, Universit~ de Bourgogne, BP 138, 21004 Dijon Cedex, France (Received 4 December 1992; accepted 21 January 1993)

Summary - Proteins of the peroxisomal membrane can be schematically divided into two groups, one being made up of more or less characterized proteins with generally unknown functions and the other consisting of enzyme activities of which the corresponding proteins have not been characterized. In the present report, these proteins and enzymes are described with the addition of unpublished results regarding their induction by peroxisome proliferators at the post-transcriptional level. Integral membrane proteins (IMPs) can be isolated using an alkaline solution of sodium carbonate. A dozen of preponderant IMPs can be seen on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the major band corresponds to a 70 kDa IMP, of which the corresponding rat cDNA is known. Some IMPs have been characterized by immunoblot analysis. Recently, a cDNA has been cloned for a peroxisome assembly factor (35 kDa IMP). Functions have also been proposed for some IMPs but are not yet firmly settled. Some IMPs (450/520, 70 and 26 kDa) are strongly induced by peroxisome proliferators. Our results extend to cipro- and fenofibrate the observation that the 70 kDa IMP mRNA level is strongly increased in di(2-ethylhexyl)phtalate-treated rats. All the enzyme activities associated with the peroxisomal membrane are involved in lipid metabolism: activation of substrates (fatty acids), ether lipid biosynthesis, and formation of precursors (fatty alcohols). It is believed that the same long-chain acyI-CoA synthetase occurs in the peroxisome as well as in the outer mitochondrial membrane and the endoplasmic reticulum. However, two highly homologous but different cDNAs encoding rat liver and brain long-chain acyI-CoA synthetases have been isolated recently. Evidence has been accumulated for a distinct synthetase that specifically activates very-long chain fatty acids. The first two steps of ether lipid biosynthesis requiere dihydroxyacetonephosphate (DHAP) acyltransferase and alkyI-DHAP synthetase, the active sites of which are located on the inner surface of the membrane. In contrast, the catalytic site of the acyl/alkyl-DHAP reductase, which generates sn-l-alkyl-glycerol-3-phosphate, is located on the outer surface. Long-chain fatty alcohols, which are obligate precursors of ether lipids and wax esters, are biosynthetized by the reduction of the corresponding acyl-CoAs via the action of an acyl-CoA reductase. Peroxisome proliferators do not appear to stimulate these enzyme activities specifically. However, we report that feno- and ciprofibrate treatments increase six-fold the palmitoylCoA synthetase mRNA level in the rat liver. peroxisomal membrane proteins / peroxisome proliferators / acyI-CoAsynthetases / ether-lipid biosynthesis / aeyI-CoAreductase

Introduction

Peroxisomes can be defined as organelles containing H202-forming oxidases and catalase [1]. Subsequent studies have shown that peroxisomes contain many other enzymes, including all the enzymes required for /3oxidation of long-chain fatty acids and those catalyzing the initial steps of ether lipid biosynthesis. All peroxisomal proteins studied thus far are synthetized on free-polysomes and imported, after translation, into existing peroxisomes [2], and new peroxisomes are formed by fission [3]. Whereas most of the so-far known peroxisomal enzymes are located inside the organelle, some enzymes, including those involved in the biosynthesis pathway of ether lipids, have been ascribed to the peroxisomal membrane. These enzyme proteins are probably minor compounds of the peroxisome membrane [4, 5]. Except for rat liver longchain acyl-CoA synthetase [6], the polypeptides responsible for these activities have not yet been characterized. In 1982, Fujiki et al [7] proposed a procedure to isolate membranes containing exclusively integral proteins (IMPs) from purified rat liver peroxisomes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis

revealed a number of major polypeptides, which have since been the subject of numerous studies. In spite of their biochemical characterization, little is known at the moment about their function. In this review, the first section will be focused on the peroxisomal IMPs; the procedures of isolation and the resulting SDS-PAGE profiles will be described. The lipid metabolism enzyme activities associated with the peroxisomal membrane will be presented in the second part. The effects of peroxisome proliferators on the peroxisomal membrane proteins and enzymes will also be mentioned. We have chosen to concentrate on mammalian peroxisomes and have deliberately omitted many excellent papers concerning peroxisomes from other organisms. We have also considered that areas such as peroxisomal diseases [8] or biogenesis of peroxisomes [9] were beyond the scope of this review.

P e r o x i s o m a l m e m b r a n e proteins

Characterization of peroxisomal membrane proteins Procedures of isolation

* Correspondence and reprints

In most studies of the peroxisomal membrane proteins, the procedure with sodium carbonate developed by Fujiki et al [7] has been performed to obtain IMPs. The carbonate

90

c Causeret et al

procedure distinguishes between IMPs, containing nonpolar sequences that are hydrophobically bound to the lipid bilayer, and peripheral membrane proteins. Fujiki et al [7] found the percentage of IMPs to be 12% of total peroxisomal proteins in rat liver, but higher values of 22 to 26% in mouse [10] and rat [11] liver have also been reported. These discrepancies may have resulted from differences in the level of released peripheral proteins during the preparation of membranes. Indeed, contamination by peripheral proteins can be high because the IMPs/total protein ratio in peroxisomes is much smaller than that in other organelles such as mitochondria or microsomes. Probably because carbonate treatment and centrifugation of purified peroxisomes yield a pellet of open-membrane sheets [7], activities of peroxisomal membrane-bound enzymes, such as acyl-CoA synthetase, dihydroxyacetone phosphate acyltransferase (DHAP-AT) and alkyI-DHAP synthase, are lost completely [11]. A phase separation of IMPs in Triton X-114 solution [12] has been presented as an alternative method to isolate IMPs from peroxisomes with the advantage of retaining the activity of most membrane-bound enzymes (except DHAP-AT) [11]. However, IMPs are not completely separated from matrix and peripheral membrane proteins, as revealed by SDS-PAGE [11, 13]. The method for the isolation of peroxisomal membranes described by Leighton et al [14] utilizes an osmotic shock in pyrophosphate buffer at pH 9.0. This procedure produces closed-membrane sheets (ghosts), which retain the peripheral membrane proteins as well as part of the inner contents [15]. Indeed, SDS-PAGE profiles of membrane polypeptides obtained by this method show some similarities with those of total peroxisomal polypeptides (see fig 1, lanes P and Mp). In contrast to the carbonate procedure, the pyrophosphate procedure along with a specific detergent treatment allows the activity of solubilized enzymes to be maintained. So, when a centrifugation pellet of pyrophosphate-treated peroxisomes is solubilized with detergents such as cholate or CHAPS and then centrifuged, > 90% of the activity of DHAP-AT was found in the soluble fraction [4, 16, 17]. Van Veldhoven et al [18] have observed that further sonication in hypotonic pyrophosphate buffer releases the matrix proteins and most of the peripheral membrane proteins. In our studies, mild sonication in pH 9.0 pyrophosphate buffer that retains the total activity of DHAP-AT, followed by centrifugation and CHAPS solubilization of the pellet, did not s~gmficantly alter the SDS-PAGE profile of membrane peroxisomal polypeptides (Causeret et al, unpublished data). Other procedures intented to diassemble peroxisomal membranes, including freezing and thawing plus mild or vigorous sonication [19] and treatments with hypo/hypertonic buffers combined with sonication and Triton X-100 solubilization [18], have also been described.

S D S - P A G E profiles

Figure 1 shows the SDS-PAGE profiles of polypeptides obtained following the carbonate or the pyrophosphate procedure from purified peroxisomes of control and fenofibrate-treated rat liver (Causeret et al, unpublished data). When ghosts from pyrophosphate-treated peroxisomes were solubilized with CHAPS, the SDS-PAGE profiles (Mp) were fairly similar to those of total peroxisomal protein (P). However, urate oxidase was completly lacking because it was not solubilized by C H A P S and was therefore sedimented upon centrifugation following solubilization (fig 1, lanes Ip). The matrix proteins, such as catalase and the/3-oxidation enzymes, were more or less released in the soluble fraction (Sp) obtained after pyrophosphate treatment and centrifugation. In contrast, the carbonate treatment followed by centrifugation essentially retained IPMs in the pellet (fig 1, lanes Mc). These electrophoretic profiles (Mc) show some of the prominent bands (corresponding to polypeptides of 69/70, 68, 41/42, 36, 28, 26, 22 and 15 kDa) generally observed by other authors (see table I). The carbonate procedure appears to be efficient in releasing peripheral membranes and matrix proteins because major peroxisomal proteins, such as catalase and urate oxidase, were almost exclusively found in the soluble supernatant fraction (fig 1, lanes Sc). In fact, the choice of the procedure to obtain membrane proteins depends on the study under investigation. The carbonate procedure provides useful information on the IMPs involved in the organization and biogenesis of the peroxisomal membrane while the pyrophosphate treatment is more suitable to analyze membrane-bound enzymes involved in the lipid metabolism.

Characteristics and f u n c t i o n s o f I M P s

Table I lists the IMPs of which the apparent Mrs have been determined by SDS-PAGE by different authors and displays some of their characteristics. The enzymes bound to the peroxisomal membrane are not listed in table I but will be discussed in another section. All the IPMs presented in table I have specifically been found in peroxisomes, except 36 kDa and 15 kDa IMPs, which are also localized in other organelles [7, 20], and perhaps some rat liver peroxisomal polypeptides recently detected in membrane preparations as being ATPases o r / a n d GTP-binding proteins. I M P 70

A 69/70 kDa IMP has been detected in peroxisomal membranes from rat [7, 21-27], mouse (named IMP 68 but similar to IMP 70) [28], and human [29, 30] liver, human fibroblasts [31-34] and Chinese hamster ovary (CHO)

Fig 1. SDS-PAGE analysis of intact peroxisomes, peroxisomal membranes and soluble fractions of control and fenofibrate-treated rats. Wistar rats were fed a standard diet supplemented with 0.3% fenofibrate for 2 weeks. Peroxisomes were purified by ultracentrifugation in a Nycodenz gradient from liver L fractions. Equal amounts of protein (50 gg) from each fraction were applied to a 10% SDS-PAGE gel, and the gel was stained with 0. I °70 Coomassie brilliant blue. P, intact rat liver peroxisomes; Mc, peroxisomal membranes prepared by the carbonate procedure [7]; Mp, peroxisomal membranes prepared by the pyrophosphate method [14] to obtain ghosts, which were then solubilized in 15 mM CHAPS [17]; Sc, soluble peroxisomal proteins of the supernatant obtained by centrifugation of carbonate-treated peroxisomes; Sp, soluble peroxisomal proteins of the supernatant obtained by pyrophosphatetreated peroxisomes; Ip, insoluble material obtained after CHAPS-solubilization of pyrophosphate-treated peroxisomal membranes and centrifugation; MM, molecular mass standards: rabbit muscle phosphorylase/3 (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), bovine erythrocyte carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and bovine milk o¢-lactalbumin (14.4 kDa).

Proteins and enzymes of the peroxisomal membrane in mammals

91

CONTROL

MM

P

Mc

Mp

Sc

Sp

Ip

kDa

94 67 catalase 43 urate oxidase

30

20.1 - 14.4

FENOFIBRATE

MM

P

Mc

Mp

Sc

Sp

Ip

kDa 94 67 catalase 43

30

20.1 _ _ 14.4

urate oxidase

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c Causeret et al

Table I. Integral membrane proteins (IMPs) in mammalian peroxisbmes. Constituent a

Source

Sensitivity to exogenous proteases

lmmunoblot analysis b

Cloned cDNA

Inducibility by proliferators c

520 450 69/70

RL RL RL, HL, HF ZSF

+ + + + + + +

no no yes

no no yes

+ + + + + + + + +

68 63 57 53 36 35

ML RIH RL HL, HF, ZSF RL CHO, HF

ND + ++ + + + ND 0 + ++

no no yes yes yes yes

no no no no no yes

+ 0 + +

29 27 25 26

RL RL RL RL

++ + + + + + + + ND

no no no yes

no no no no

+ + + + + +

22

RL, ML, HL HF, ZSF

+

yes

no

+

15

RL

ND

no

no

ND

+ + +

Proposed function

References

ATPase 48 ATPase ATP-binding 7, 20-27, 29-34, transport protein 38, 39, 58 (ATPase?) unknown 28, 36 unknown 54 unknown 55 unknown 31, 32 unknown 20-22, 24 peroxisome assembly 58, 59 factor (PAF 1) GTP-binding protein 56 GTP-binding protein 56 GTP-binding protein 56 unknown 17, 20-23, 25-27 (Causeret et al, unpublished data) porin 7, 10, 18, 19-21, 23-25, 31, 32, 34, 49, 50, 53 unknown 7, 20, 21

aSubunit size (kDa) was determined by SDS-PAGE stained with Coomassie brilliant blue. In some cases, polypeptides were visualized by autoradiography of the gel (63 kDa) or by immunoblotting (25, 27 and 29 kDa). The apparent molecular masses of 520 and 450 kDa have been estimated by gel filtration chromatography. bMost of the antibodies against IMPs were monospecific or polyspecific polyclonal antisera obtained by immunization of rabbits with partially purified or non purified proteins from rat liver peroxisomal membranes. The 57 kDa polypeptide has been identified using a monoclonal antibody [55]. The antibody against the 35 kDa polypeptide has been obtained by rabbit immunization with a synthetic peptide [58]. CRat liver peroxisome proliferators used were hypolipidemic drugs such as clofibrate, ciprofibrate, fenofibrate, BM 15766, and nafenopin or plasticizers such as di(2-ethylhexyl)phthalate (DEPH). Abbreviations: RL, rat liver; RIH, rat isolated hepatocytes; ML, mouse liver; HL, human liver; HF, human fibroblasts; ZSF, Zelleweger syndrome fibroblasts; CHO, Chinese hamster ovary cells. ND, not determined.

cells [35]. This protein generally forms the major band in SDS-PAGE profiles. Polypeptides with apparent Mrs of 68, 41/42, 28 kDa have been shown to derive from the 69/70 kDa IMP by an endogenous proteolyse process during purification of peroxisomes [21-24]. The sensitivity of the 69/70 kDa IMP to endogenous proteases may be one of the poss~bd~tles put forward to explain the near absence of this protein in the SDS-PAGE profiles shown in figure 1 (lanes Mc). Moreover, a 70-kDa protein isolated from peroxisomal membranes of mouse liver [10, 36] has revealed to be different from the rat liver 69/70-kDa IMP and, in fact, to be enoyl-CoA hydratase/hydroxyacyl-CoA dehydrogenase [37], a poorly soluble matrix component partly retained in the membrane fraction after carbonate extraction [13, 19]. cDNA clones for the 69/70 kDa IMP have been isolated from rat [38] and human [39] liver and sequenced. The predicted amino acid sequence corresponds to a 75.3-kDa protein (however, still called IMP 70) and shows a carboxyl terminal sequence with strong similarities to that of a superfamily of ATP-binding proteins, most of which are involved in membrane transport. The SKL motif, a targeting signal for protein import into peroxisomes, is not present at the C-terminus of the IMP 70 [38, 39]. From limited proteolysis, the ATP-binding domain o f rat IMP 70 has been found to be exposed on the cytosolic face as a 24-kDa fragment [38]. The authors suggested

that IMP 70 may be involved in active transport for proteins a n d / o r substrates across the peroxisomal membrane. This transport function proposed for IMP 70 may be connected to the observation that the import of acyl-CoA oxidase into rat liver peroxisomes is ATP-dependent [40]. Moreover, participation of an ATPase in the transport of substrates across the peroxisomal membrane has been reported for D H A P - A T activity because the reaction requires ATP [41]. Oligomycin-resistant ATPase activity has been detected on the cytosolic face of the peroxisomal membrane in rat liver [42], but the polypeptide(s) responsible for this activity have not been identified. Presence of ATPase activity(ies) in rat liver peroxisomes has also been reported by other authors [43-46], but roles and functions require clarification. In spite of the sequence homology of IMP 70 with a family of membrane transport ATPases, including the eukaryotic P-glycoprotein associated with multidrug resistance in humans [47], at the moment, no ATPase activity has been demonstrated for IMP 70. Recently, Shimizu et al [48] have described the characteristics of two types of ATPase, N-ethylmaleimidesensitive and -resistant ATPases, in the peroxisomal membrane of rat liver. These two ATPases were partially separated by gel filtration chromatography, providing apparent MrS of 520 kDa (N-ethylmaleimide-sensitive enzyme) and 450 kDa (N-ethylmaleimide-resistant enzyme).

Proteins and enzymesof the peroxisomal membrane in mammals I M P 22

A peroxisomal 22-kDa IMP (IMP 22) has been detected in rat [7, 18, 19-27, 47], mouse (but only as a minor constituent) [10], and human [49, 50] liver and in human fibroblasts [31, 32, 34, 49, 50]. Van Veldhoven et al [18] have suggested that IMP 22 is responsible for the poreforming activity of the peroxisomal membrane in rat liver. A conductance channel has been described in the peroxisomal membrane [51, 52], but no IMP was yet identified for this role, although involvement of IMP 22 is possible. In vitro translation studies have shown that the IMP 22, like the 69/70, 36 and 26 kDa proteins, is synthetized on free polysomes as a mature protein [24, 25, 53]. IMP 22 contains 44% hydrophobic amino acids, which is consistent with the features of an IMP [53]. Mild trypsinization of isolated peroxisomes causes the disappearance of IMP 22 with the concomitant appearance of an only slightly smaller polypeptide (21 kDa) [53]. This indicates that a large portion of the polypeptide is inserted into the membrane lipid bilayer. Other I M P s

Several other polypeptides of the peroxisomal membrane, 63 kDa [54], 57 kDa [55], 36 kDa [20-22], 26 kDa ([17, 20-23, 25-27]; Causeret et al, unpublished data) and 15 kDa [7, 20, 21] polypeptides, have been observed in rodent liver, but their function remains to the elucidated. Recently, the presence of three small GTP-binding proteins of 29, 27 and 25 kDa has been detected in the peroxisomal membrane [56]. The authors suggested that these proteins may be involved in peroxisome biogenesis, but further studies are necessary to establish this proposal. I M P s in peroxisomal disorders An impairment of peroxisomal functions is encountered in the Zellweger syndrome and in other related human diseases. The cells (skin fibroblasts) and tissues (liver) of these patients are characterized by the virtual absence of morphologically distinguishable peroxisomes, probably due to a primary defect in the biogenesis of peroxisomes. The question arises as to the fate of the IMPs in cells deficient in peroxisomes. IMP 22 [25, 31, 32, 49, 50, 57] and IMP 70, [25, 31, 32, 49] and other IMPs (36, 53 and 140 kDa) [31, 32, 49] have been detected in Zellweger patients in normal amounts or more often in more or less low amounts. In fact, in these patients several lines of evidence have been presented for the presence of aberrant, empty membrane structures (peroxisome ghosts), containing the abovementioned IMPs [31-33, 49, 50]. Moreover, normal synthesis of IMP 70 without further degradation has been evidenced in CHO cell mutants deficient in peroxisomes [35]. In spite of the presence of some major IMPs in cells lacking normal peroxisomes, it has been speculated that IMP(s) may be responsible for the ineffective assembly of peroxisomes. Recently, Tsukamoto et al [58] have cloned and characterized a rat cDNA encoding a peroxisomal membrane protein of M r 35 kDa (called PAF-1) that restores the biogenesis of peroxisomes in CHO cell mutants defective in the assembly of peroxisomes. The same group of researchers [59] isolated a cDNA encoding a human PAF-1 that complemented the defect of peroxisomal structure and functions in fibroblasts of a Zellweger patient. PAF-I contains a potential zinc- and DNA-binding signature subsequence (ring finger) in its carboxyl-terminus [60].

93

Effects o f proliferators

The peroxisome proliferators are known, above all, for strongly increasing the level of/3-oxidation enzymes in the liver of rodents. Figure 1 (lanes P and Sp) shows a strong increase in the 72, 48, 23 and 42 kDa polypeptides, which can correspond to the A, B and C subunits of acyl-CoA oxidase and to thiolase, respectively, in the peroxisomes of fenofibrate-treated rat liver. Concerning the effects of proliferators on peroxisomal membrane proteins, a high specific increase in IMP 70 is generally observed in the rat liver [21-23, 25, 26, 38]. For unclear reasons, such an increase in IMP 70 could not be observed in our SDS-PAGE profiles (fig 1, lanes Mc). Kamijo et al [38] have studied the effects of proliferators on IMP 70 at the posttranscriptional level by using a fragment of IMP 70 cDNA as a probe. They have shown that di(2-ethylhexyl)phthalate (DEHP) increased the IMP 70 mRNA by more than 10-fold. This result is in agreement with that obtained by Suzuki et al [25] in in vitro translation experiments. Figure 2 shows Northern blots of IMP 70 mRNA obtained in control and fibrate-treated animals when hybridization was performed with an oligodeoxynucleotide probe specific for IMP 70 (Causeret et al, unpublished data). Both proliferators, ciprofibrate and fenofibrate, increased the IMP 70 mRNA about six- and five-fold, respectively. These results and those of Kamijo et al [38], and Suzuki et al [25] demonstrate that in the rat administration of peroxisome proliferators specifically induces IMP 70. In the liver of mice IMP 68, which is similar to rat IMP 70, is also increased following treatment with proliferators, but its specific content in the peroxisomai membrane appears to be unchanged. The modest induction of IMP 68 in the mouse has been confirmed by means of Northern analysis, using a rat IMP 70 cDNA as a probe, and experiments of gene transcription in vitro [28]. In contrast with IMP 70, IMP 22 does not appear to be influenced markedly by the proliferators such as fenofibrate (fig 1, lanes Mc), clofibrate [22], DEHP [23, 25], or BM 15766 [26]. Moreover, figure 1 shows that a 26-kDa protein was the most induced IMP in the liver of the fenofibrate-treated rats. Similar results were obtained in cipofibrate-treated rats [17]. Induction of a 26-kDa IMP has also been observed in the liver of DEHP-treated rats by Hashimoto et al [23] and Suzuki et al [25]. Other peroxisomal polypeptides, described as IMPs in table I, including 520 and 450 kDa ATPases [48] and a 57 kDa IMP [55], have been reported to be induced by clofibrate. Peroxisomal membrane-bound enzymes

All the enzyme activities so far described as being associated with the peroxisomal membrane are involved in lipid metabolism. Before fatty acids undergo/3-oxidation in the peroxisomal matrix, their activation to the corresponding CoA-esters through acyl-CoA synthetases is believed to take place at the site of the peroxisomal membrane. Moreover, figure 3 shows that all the enzymes (long-chain acyl-CoA synthetase, acyl-CoA reductase, dihydroxyacetone-phosphate (DHAP) acyltransferase, alkyI-DHAP synthase, and acyl/alkyl-DHAP reductase) required for the onset of the biosynthesis of ether lipids from fatty acids, DHAP, and cofactors are present in peroxisomes in membrane-bound form. The enzymes that catalyse the biosynthesis of wax esters are possibly associated with the membrane of the peroxisome, and, therefore, will also be discussed in the present review. Lastly, available informa-

A

Control Ciprofibrate Fenofibrate

28__ IMP 70 18m

B

Control Ciprofibrate Fenofibrate

28__ Acyl-CoA synthetase 18__

C

Control

Ciprofibrate

Fenofibrate

28m

18m

Tubuline

Fig 2. Effects of ciprofibrate and fenofibrate on hepatic IMP 70 and long-chain acyl-CoA synthetase mRNAs in rats: Northern blot analysis. Northern blots of total rat liver RNA (20 fzg/well) electrophoresed, transfered to nylon filters, and then probed with 32p_ end labeled oligonucleotides specific for the mRNAs encoding IMPT0 (A), long-chain acyl-CoA synthetase (B), and ~t-tubulin (C). The following oligonucleotides were used: 5'-TAG-TAC-TCG-TGG-TGT-TTC-CA-3' corresponding to the complement of nucleotides 1888-1907 of the rat liver IMP 70 cDNA [38]; 5'-GGG-TGC-ACA-GCA-ATG-CC-3' corresponding to the complement of nucleotides 1966-1982 of the rat liver long-chain acyl-CoA synthetase cDNA [6]; 5'-GAC-ATC-TTT-GGG-GAC-CAC-ATC-ACC-ACG-3' corresponding to the complement of the nucleotides 39-66 of the rat ~t-tubulin 3'-end cDNA clone PT25 [212]. The method used was essentially as described by Waxman [213] and modified by Causeret et al (unpublished data).

Proteins and enzymesof the peroxisomal membrane in mammals Verylong chain FA

Longchain FA

AcyI-CoA

Acyl-CoA

/5-OXl DATI0 N Fatty alcohols d-DHAP

/ / /Alky.i.G3P ~

ENDOPLASMIC

RETq~LUM \

-~.

~'~'~AlkyI'DHAP

~

I ~

~

~

AlkyI-DHAP

Fig 3. Topography of membrane-bound enzymesin peroxisomes. 1, Palmitoyl-CoA synthetase: 2, acyl-CoA reductase; 3, DHAP acyltransferase; 4, alkyI-DHAP synthase; 5, acyl/alkyI-DHAP reductase; 6, lignoceroyI-CoAsynthetase. DHAP, dihydroxyacetone phosphate; G3P, sn-glycerol-3-phosphate;FA, fatty acids. Modified from [131].

tion on the effects of peroxisome proliferating hypolipidemic drugs on the peroxisomal membrane-bound enzyme activities will be reported.

Activation of fatty acids Long-chain acyI-CoA synthetases Fatty acids are first activated in the presence of ATP and Mg2+ to their corresponding acyl-CoA esters, before being incorporated into glycerolipids or degraded by /3oxidation. Mammalian cells contain a variety of different acyl-CoA synthetases (or ligases), of which the long-chain acyl-CoA synthetase (or palmitoyl-CoA synthetase) (E.C. 6.2.1.3) has been characterized in most detail. Long-chain acyi-CoA synthetase activates a variety of fatty acids, including saturated Cm-C~s and unsaturated C16-C20 fatty acids, but is relatively inefficient with very-long-chain fatty acids such as lignoceric acid [61-68]. Long-chain acyl-CoA synthetase has been purified to homogeneity from rat liver by Tanaka et al [63], with an apparent M r of approximately 76 kDa. The enzyme, which occurs in the outer mitochondrial membrane and the membrane of the endoplasmic reticulum, is also present in peroxisomes [62, 69-73]. Peroxisomal long-chain acyl-CoA synthetase is a membrane-bound protein, the enzymatic site of which is located on the cytosolic surface [70, 74, 75]. This peroxisomal enzyme is generally thought to be identical to that present in mitochondria and microsomes, as judged by several physicochemical, catalytic and immunological properties of the enzymes purified from different subcellular fractions [63, 76] and by the sequence analysis of cDNAs [6].

95

Recently, a cDNA encoding rat long-chain acyl-CoA synthetase, predicted to contain 699 amino acids (78 kDa), was isolated from a library prepared from rat liver poly(A) ÷ RNA [6]. The pattern of expression of the long-chain acylCoA synthetase mRNA was tissue-specific because the level of expression in brain, lung, and small intestine was only 10°70 of that in liver, heart and epididymal adipose tissue [6]. The low level of expression in the brain, which contains much more lipid than most of the other tissues in mammals, has prompted these authors to screen a rat brain cDNA library with a long restriction fragment from the rat liver long-chain acyl-CoA synthetase cDNA using a low stringency condition [68]. A novel long-chain acyl-CoA synthetase was found, which consists of 697 amino acids, has 65% identity with the rat liver enzyme, and is expressed predominantly in brain [68]. The discovery in the same species of two distinct enzymes, highly homologous in their sequence and similar in their substrate specificity [68], indicates that the identity of long-chain acyl-CoA synthetases in microsomes, mitochondria and peroxisomes may be questioned, as already suggested by the results from Singh and Poulos [77]. Interestingly, both rat liver and brain longchain acyl-CoA synthetases are lacking in the SKL motif for peroxisomal targeting [6, 68], as is the 70 kDa ATP-binding protein, another peroxisomal membrane protein [38, 39]. An acyl-CoA synthetase, called arachidonoyl-CoA synthetase, specific for arachidonate and other eicosanoid precursor fatty acids, has been described in a variety of cells and tissues [78-83]. The results suggested that nonspecific long-chain acyl-CoA synthetase, which also utlizes polyunsaturated fatty acids as substrate, and arachidonoylCoA synthetase represent two distinct enzymes. However, an opposite conclusion has recently been drawn in studies regarding human platelets [66, 67]. Presence of arachidonoyl-CoA synthetase activity in peroxisomes, at the present time, remains unexplored. Apart from their ability to catalyse the well-known/3oxidative chain shortening of long-chain fatty acids, peroxisomes appear to be indispensable for the oxidation of verylong-chain fatty acids, pristanic acid, di- and trihydroxycoprostanic acids, dicarboxylic acids and prostaglandins. Before these compounds can undergo/3-oxidation, activation to their corresponding CoA-esters must occur; activation of very-long-chain fatty acids will be discussed in the following section. With regard to pristanic acid, Wanders et al [84] have observed that pristanoyl-CoA and palmitoylCoA synthetase activities were similarly distributed among peroxisomes, mitochondria and microsomes, and concluded on the basis of a set of experiments that pristanic acid is activated by the same enzyme which activates long-chain fatty acids. If only the pristanoyl-CoA synthesized by the peroxisomal synthetase is available for/3-oxidation within the peroxisome is not known. In contrast, evidence is emerging that the other compounds would be activated only in microsomes by separate specific synthetases [85-88]. Moreover, Bronfman and his collaborators [89, 90] have reported that, in rat liver, hypolipidemic drugs such as ciprofibrate undergo activation to CoA-esters and presented results suggesting that the peroxisome proliferators of the fibrate series are activated by the non-specific longchain acyl-CoA synthetase. However, the possible role of the peroxisomal enzyme was not clarified [91]

Very-long-chain acyI-CoA synthetase Very-long-chain fatty-acids, such as lignoceric (24:0) or cerotic (26:0) acids, are preferentially, and possibly exclu-

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c Causeret et al

sively, oxidized in peroxisomes [92-95]. These fatty acids have to be converted into acyl-CoA esters before being metabolized, and very-long-chain acyl-CoA synthetase (or lignoceroyl-CoA synthetase) activities, dependent on ATP and Mg 2+ [96], have been found to be present in peroxisomes [77, 97-100], as well as in microsomes, but not in mitochondria (except in [101 ]). Current evidence suggests that it is only the CoA-ester synthetized by the peroxisomal enzyme which is available for fl-oxidation within the peroxisome [100, 102]. In recent years, evidence has been brought forward for the existence of a lignoceroyl-CoA synthetase distinct from the palmitoyI-CoA synthetase [74, 77, 83, 97, 99, 100, 103-107] although this has been questioned by Khishimoto and coworkers [108]. It is noteworthy that the p a t h o g n o m o n i c a c c u m u l a t i o n of very-long-chain fatty acids in X-linked adrenoleukodystrophy is due to deficiency of the peroxisomal lignoceroylCoA synthetase activity, whereas activation and oxidation of palmitic acid proceed normally in peroxisomes [99, 102, 105, 106, 109, 110]. The active site of the peroxisomal lignoceroyl-CoA synthetase would be located on the luminal surface of the peroxisomai membrane according to Lazo et al [74] (see also [73]) or, at the opposite, on the cytosolic face according to Lageweg et al [75], as observed for the microsomal lignoceroyl-CoA activity [96]. The reasons for this discrepancy have remained unclear so far. Recently, Singh et al [105] demonstrated that transport of palmitic acid through the peroxisomal membrane requires prior synthesis of palmitoyl-CoA by palmitoyl-CoA synthetase on the cytoplasmic surface of peroxisomes, whereas lignoceric acid is transported as such before being activated by lignoceroyI-CoA synthetase on the luminal face. The same authors [105] suggested that the selective ATPindependent transport of palmitoyl-CoA and lignoceric acid, but not of palmitic acid and lignoceroyl-CoA, may be mediated by different specific peroxisomal membrane proteins, distinct from pore-forming proteins associated with non-specific permeability of small hydrophilic molecules through the peroxisomal membrane [18] and from ATP-dependent translocases for peroxisomal protein import [40]. Biosynthesis o f ether lipids Overview The key enzymes which catalyze the biosynthesis of triacylglycerols and phosphoglycerides are virtually absent in peroxisome~, except those required for the onset of the ether lipid biosynthesis [72, 111, 112]:

Dihydroxyacetone-phosphate

(DHAP) DHAP acyhransferase (EC 2.3.1.42) AcyI-DHAP Fatty a l c o h o l ~ AlkyI-DHAP synthase (EC 2.5.1.26) Fatty acid ~

q¢ AlkyI-DHAP

NADPH+H÷~ NADP* < sn- I -A Ikyl-glycerol-3-phos phate (AlkyI-G3P)

Acyl/alkyI-DHAP oxidoreductase (EC 1.1.1.101)

The fatty alcohols, precursors of the ether bond along with acyi-DHAP, are formed by reduction of acyl-CoAs, and the reaction is catalyzed by an acyl-CoA reductase (long-chain fatty alcohol forming) also present in peroxisomes. The synthetized alkyi-G3P is transported to the endoplasmic reticulum, which contains the enzymes required to complete the biosynthesis of glycero-ether lipids [113]. AlkyI-G3P may freely diffuse from the peroxisome to the endoplasmic reticulum, ie take place without the involvement of a specific carrier protein [114], but this is still not firmly established [115, 116]. Such transport could be facilitated by structural membrane associations between both organelles [117]. The data summarized above indicate that peroxisomes have a specialized role in the synthesis of the ether-linked lipids [118]. Dihydroxyacetone-phosphate acyltransferase ( D H A P - A T)

Although microsomal glycerol-3-phosphate acyltransferase (G3P-AT) has a dual catalytic activity (G3P-AT and DHAP-AT activities) [119, 120], the main function of the acyI-DHAP pathway lies in the synthesis of ether lipids, which have acyI-DHAP as an obligate precursor. Indeed, cellular DHAP-AT activity has been shown to be almost exclusively localized in the peroxisome fraction of different cells and tissues, including guinea pig [11 I, 121 - 123], rat [72, 111,124, 125] and mouse [126] liver, rat brain [101, 111, 127], rat [126] and mouse [128, 129] kidney, guinea pig intestinal mucosa [130, 131], human skin fibroblasts [94], preadipocytes 3T3-L 1 [132], and hepatoblastoma ceils HepG2 [133]. The enzyme G3P-AT is virtually absent from peroxisomes [111]. The peroxisomal localization of DAHP-AT has been confirmed by the finding that the enzyme is strongly deficient in tissues and cells from Zeliweger patients, which do not contain morphologically detectable peroxisomes, whereas G3P-AT activity is almost normal [134-136]. Peroxisomal DHAP-AT is an enzyme distinct of microsomal G3P-AT because: 1) partially purified DHAP-AT has negligible G3P-AT activity [16]; 2) properties of these two acyltransferases are quite different [122, 137-139]; and 3) selective changes in the two activities occur during cell differentiation [214]. For example, the DHAP-AT and G3P-AT activities of the microsomal G3P-AT are inhibited by N-ethylmaleimide [119, 120], whereas the peroxisomal DHAP-AT activity is insensitive to the thiol reagent [16, 122, 140]. Furthermore, both microsomal acyltransferase activities, are maximum at a pH value of 7.5 to 8.0 [119, 120, 141, 142]. In contrast, peroxisomal DHAP-AT has a low pH optimum (5.5), and at this low pH microsomal G3P-AT is inactive [72, 121, 129, 137, 138, 142-145]. In the peroxisome, DHAP-AT is present in a membranebound form, and its active site is located at the inner aspect of the membrane [75, 122, 137, 145-147]. The enzyme appears not to be very specific because relatively high rates of acylation were obtained with Ci2-C20 saturated and Cl8 unsaturated fatty acids (but not lignoceric acid) as substrate in the presence of CoA, ATP and Mg 2÷ [148]. The enzyme has been purified from guinea pig liver peroxisomes, first partially [16] and then to homogeneity [4, 215]. A M r of 68 kDa was assigned to the enzyme on SDS-PAGE but its sequence remains unknown. Recently, Zoeller et al [149] have isolated plasmaiogen-deficient mutants in a murine macrophage-like cell line, which were deficient in DHAP-AT but contained intact, functional peroxisomes. The deficiency in the DHAP-AT activity was the unique lesion in the ether lipid biosynthetic pathway. Transfection of these DHAP-AT defective mutants with

Proteins and enzymesof the peroxisomal membrane in mammals a cDNA library and selection of revertant cells could allow a DHAP-AT cDNA to be cloned, as has been done by Tsukamoto et al [58] for IMP 35 (PAF-I). A Ikyl-DHA P synthase

Although the product (acyI-DHAP) of the reaction catalyzed by DHAP-AT is transported very fast across the membrane and can diffuse to the endoplasmic reticulum, it is also utilized at a high efficiency as substrate by alkylDHAP synthase, suggesting a close interaction of the two enzymes in the peroxisomal membrane [115, 116]. Indeed, aikyI-DHAP synthase has generally been found to be colocalized with DHAP-AT in peroxisome fractions on centrifugation [77, 101, 111, 123, 130, 132, 142, 150, 151]. As has been observed for DHAP-AT, lack of peroxisomes in some inherited diseases also leads to a deficiency in alkyI-DHAP synthase activity [135, 152-155]. The enzyme is associated with the peroxisomal membrane, and its catalytic site is located on the inner surface of the membrane [145, 147, 156]. The peculiar molecular mechanisms of the formation of the ether bond have been investigated in detail using postmitochondrial fractions [157, 158] or partially purified enzyme [159-163]. AlkyI-DHAP synthase accepts a wide variety of primary alcohols as substrate, comprising C12-C~8 saturated and C~8 unsaturated chains [123, 158, 164, 165] (see also [166]). With regard to the chain length of the acyl group in the acyl-DHAP species utilized as substrate, palmitoyl-DHAP has proven more active than stearoyl- and myristoyI-DHAP [158]. To our knowledge, alkyI-DHAP synthase has not yet been purified to homogeneity. A c y l / a l k y I - D H A P reductase

A single enzyme, acyl/alkyl-DHAP reductase, which has high affinity for NADPH, catalyzes the stereospecific reduction of acyI-DHAP to sn-l-acyl-G3P and that of alkylDHAP to sn-l-alkyl-G3P [167]. Acyl/alkyI-DHAP reductase activity is present both in peroxisomes and in the endoplasmic reticulum [72, 111, 123, 124, 142, 168], but not in mitochondria. Whether or not the same enzyme is present in both subcellular compartments is not known. From the lability of the enzyme toward trypsin in intact peroxisomes (and also in microsomes), it was concluded that acyl/alkylDHAP reductase is present on the cytosolic side of the membrane of these organelles [131, 147, 168]. Unlike DHAP-AT and alkyI-DHAP synthase, the reductase activity is normal in the cells of Zellweger patients [135, 136], suggesting that all of the reductase may be targeted to the endoplasmic reticulum in the absence of peroxisomes. Since acyI-DHAP and alkyi-DHAP synthetized inside the peroxisome can probably be transported fast across the peroxisomal membrane [115, 116], both can be enzymatically reduced to the corresponding glycerolipids by cytosolic NADPH. Guinea pig liver acyl/alkyl-DHAP reductase has been solubilized and purified partially [169, 170] and then to homogeneity by Datta et al [5]; it would constitute approximately 3°70 of total peroxisomal membrane-bound proteins. The data on SDS-PAGE and size exclusion chromatography together indicate that the native enzyme would be a monomer of 60 kDa [5]. To our knowledge, no report concerning generation of a specific antibody against the purified enzyme or obtention of a cDNA has been issued. A cyl-CoA reductase

Long-chain alcohol, which is the substrate for alkyI-DHAP synthase, is an obligate precursor of ether lipids in animals [112, 167]. Although exogenous long-chain alcohols

97

(for example, those derivated from skin surface lipids ingested by licking in the rat [171]) are absorbed and readily taken up by the tissues, these alcohols are mainly oxidized to fatty acids by microsomal oxidases [172, 173] and are therefore poorly utilized for ether lipid biosynthesis [172, 174]. In fact, the ether lipid precursor alcohols are primarily biosynthesized in situ by reduction of the corresponding fatty acyl-CoAs [175, 176]. Evidence has been presented that a single enzyme, acyl-CoA reductase (longchain alcohol forming), probably catalyses the two reduction steps (acyl-CoA --, aldehyde --, alcohol) without release of the intermediate aldehyde [172, 176, 177]. NADPH is the specific coenzyme, which cannot be replaced by NADH [175-179]. The reductase is present as a membrane-bound enzyme in different mammalian tissues, including rabbit harderian gland [180], rat brain [176], mouse preputial gland [181] and bovine meibomian gland [177], and is primarily localized in peroxisomes [131, 182]. Sensitivity to trypsin digestion indicates that the reductase is on the outer face of the peroxisomal membrane [131, 180, 181], so that cytosolic NADPH can be utilized for the reduction of acyl-CoAs to fatty alcohols. In fibroblasts from Zellweger syndrome patients the acyl-CoA reductase, like the acyl/alkyl-DHAP reductase, which both are localized on the cytosolic surface of the peroxisomal membrane, is not affected by the absence of peroxisomes characteristic of the disease [135, 136]. The acyl-CoA reductase has been found to be much less active than the other peroxisomal enzymes involved in the ether lipid biosynthesis pathway (DHAP-AT, alkyI-DHAP synthase and acyl/alkyl-DHAP reductase) when the different activities were assessed in homogenates of cultured human fibroblasts [136] and in purified peroxisomes of guinea pig intestine mucosal cells [131]. Moreover, long chain acyl-CoA synthetase appears to be more active than DHAP-AT, alkyI-DHAP synthase and acyl/alkyl-DHAP reductase, in peroxisomes from rat liver [72, 75, 77] and brain [101]. Although it is not possible to say with certainty what is occurring in the living cell, it may be postulated that the reduction of acyl-CoAs is the rate-limiting reaction for the overall biosynthesis of ether lipids in animal tissues. The reductase also controls, at least in part, the alkyl (and alk-l'-enyl) chain length distribution at the sn-1 position of ether lipids, of which the composition of the ether chains is simple in most mammalian tissues and consists almost entirely of C 16:0, C 18:0 and C 18:1 groups. Indeed, the enzyme is fairly specific for palmitoyl-, stearoyl- and oleoyl-CoA [164, 176, 178, 183], whereas long-chain acyl-CoA synthetase [61-65, 68] and alkylDHAP synthase [123, 158, 164, 183] are able in vitro to utilize polyunsaturated or shorter chain fatty acids and alcohols, respectively. As far as very long-chains (> C20:0) are concerned, they are poor substrate for palmitoyl-CoA synthetase [68], acyl-CoA reductase [176], DHAP-AT [140] and alkyI-DHAP synthase [123, 158, 183], and the combined action of these enzymes is probably responsible for the exclusion of > C20:0 moieties from ether lipids. Furthermore, if the active site of lignoceroyl-CoA synthetase is really on the inner face of the peroxisomal membrane [74, 105], the synthetized very-long-chain acyl-CoAs, which are readily oxidized inside the peroxisome, may not be available for the acyl-CoA reductase located on the outer face. Biosynthesis o f wax esters

Wax esters are major components of skin surface lipids secreted by sebaceous glands. It is admitted that their

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c C a u s e r e t et al

biosynthesis proceeds from long-chain fatty acids as it follows: Fatty~/acids Long-chain /

synthetase

AcyI-CoAs

S \

acyI-CoA

15-

,~ A c y I - C o A Fatty

~-----~,L

Wax

reductase

05

alcohols F~'ttty a l c o h o l a c y l t r a n s f e r a s e

esters

It was believed that the enzymatic synthesis of wax esters takes place in mammalian microsomes [177, 184, 185]. Kolattukudy and Rogers [177] have shown microsomes from the bovine meibomian gland to contain N A D P H utilizing acyl-CoA reductase and fatty alcohol acyltransferase activities. Furthermore, in the presence of hexadecanol and different acyl-CoAs, palmitoyl- and oleoyl-CoAs gave maximal rates of esterification; these results were consistent with the fatty acid composition of meibomian wax esters [186, 187]. However, the microsome fraction used for enzyme assays in this work [177] and others [184, 185] was a postmitochondrial particulate fraction, which most probably included peroxisomes besides microsomes; the precise subcellular localization of wax ester biosynthesis remains therefore uncertain. Recently, Hardeman and Bosh [151] have shown that purified rat liver microsomes converted exogenous [~4C]hexadecanol to monoester wax, suggesting that acyltransferase activity was located in the endoplasmic reticulum. In this study, exogenous [InC]palmitic acid was esterified to form monoester wax only in the presence of exogenously added hexadecanol, suggesting that reductase activity was not located in microsomes, in spite of the bimodal localization of acyl-CoA reductase (both in microsomes and peroxisomes) observed by other authors [72, 111]. Moreover, ultrastructure of sebaceous gland cells in mammals [188-190] and birds [191] displays close associations between peroxisomes and the endoplasmic reticulum. Furthermore, although direct membrane continuities have never been observed, electron dense crossbridges between the two compartments have been described [188, 191]. Ultrastructural, intermembraneous relationships between bothsorganelles are also visible in kidney cells [117, 192], enterocytes [193], hepatocytes [194-197], and adipocytes [198]. Together, these observations and the biochemical data imply a functional role of the peroxisome-endoplasmic reticulum associations in the biosynthesis of wax esters. Reduction of acyl-CoAs to fatty alcohols may occur on the cytosolic side of peroxisomes and esterification may proceed in the microsomal membrane. Therefore, free fatty alcohols would have to be transported from peroxisomes to the endoplasmic reticulum. Once synthesized, wax esters accumulate into the lumen of microsomal membranes to form fat bodies before secretion. Precise localization of the enzymes involved in wax ester biosynthesis remains to be settled.

Effects of peroxisome proliferators Ether-lipid biosynthesis enzymes A number of hypolipidemic drugs, which are structurally related (such as fenofibrate and ciprofibrate) or unrelated to clofibrate, and other compounds such as D E P H ,

0

4)

2

4

6

[

]

I

[ Treatment

Weeks

Fig 4. Effects of ciprofibrate and fenofibrate treatments on specific activities of DHAP-AT in purified rat liver peroxisomes. Control rats ( 5 ) were fed standard diet. Rats were fed a standard diet supplemented with 0.02°7o ciprofibrate (A) or 0.307o fenofibrate (O) during 2 week periods. DHAP-AT activity was measured at pH 5.5, essentially as described by Schutgens et al [134, 144]. The data were drawn from [17].

induce a proliferation of peroxisomes in rat liver accompanied by hepatomegaly and increased peroxisomal /3oxidation (see fig 1). The peroxisomal membrane-bound enzymes, which are involved in lipid metabolism, have not been examined much for their response to these agents, with the exception of D H A P - A T . Following administration of hypolipidemic drugs, 1.2- to 2.0-fold increases in the specific activity of D H A P - A T have generally been observed in homogenates or in L fractions of rat [17, 111, 199-202] and mouse [203] liver and embryonic cells [204]. However, figure 4 shows that no significant increase occurs when the D H A P - A T activity is examined in purified peroxisomes of fibrate-treated rat liver. Absence of stimulation by proliferators of the DHAP-AT in purified peroxisomes has also been observed by other authors [17, 199, 200, 205,206]. Furthermore, Causeret et al [17] have found that, when DHAP-AT was solubilized from rat liver peroxisomal membranes, the specific activity of the enzyme was not altered by fibrate treatments. It can therefore be concluded that peroxisome proliferators do not specifically induce DHAP-AT. Interestingly, Herrera et al [207] have reported that clofibrate stimulates the specific activity of cytosolic G3P-dehydrogenase in rat liver, providing DHAP which is believed to be freely transported through the peroxisomal membrane [208]. In contrast with the in vivo effects of the hypolipidemic drugs, these proliferators strongly inhibit peroxisomal D H A P - A T in vitro [209]. Concerning the other peroxisomal enzymes involved in the ether-lipid biosynthesis, inconsistent changes have been found with regard to the influence of proliferators on acyl/alkyl-DHAP reductase [168,202] and alkyI-DHAP synthetase [202] activities in rodent liver. At the moment, no data are available regarding acyl-CoA reductase. In conclusion, the ether-lipid biosynthesis enzymes appear to be not much altered by peroxisome proliferators, in contrast to some IMPs such as IMP 70. Changes, when observed, may not be related to the specific action of proliferators at the gene level, but may be a consequence of the proliferation of peroxisomal membranes.

Palmitoyl-CoA synthetase Palmitoyl-CoA synthetase activity is mainly distributed in the mitochondria and the endoplasmic reticulum. Krisans

Proteins and enzymes of the peroxisomal membrane in mammals et al [69] found that, in normal rat liver, 70./0 of the total

enzyme activity was located in peroxisomes. These authors [69] also reported that clofibrate treatment of rats caused 2.6- and 3.9-fold increases in the palmitoyl-CoA synthetase activity measured in homogenates and L fractions respectively. They calculated that the clofibrate-treated rat peroxisomes had 12°/0 of the total cell palmitoyI-C.oA synthetase activity (ie twice the control value), suggesting a specific stimulation of the peroxisomal enzyme. The Northern blots shown in figure 1 exhibit a strong increase (4.9- and 6.6-fold) in the rat liver palmitoyl-CoA synthetase m R N A level upon fenofibrate and ciprofibrate treatments, respectively. This Northern blot analysis indicates that the stimulation by fibrates of the rat liver palmitoylCoA synthetase occurs at a pre-translational level. In our work, whether the oligonucleotide probe was specific for a palmitoyI-CoA synthetase located in a definite organelle could not be specified (see [6]). In contrast to the action of fibrates, feeding rats a high fat diet that causes a considerable proliferation of peroxisomes with an increased capacity of peroxisomal/3-oxidation appears to increase the peroxisomat palmitoyI-CoA synthetase activity no more than the catalase activity that is only two-fold enhanced [210].

Conclusion and prospects The rapid advances in our understanding of peroxisome biogenesis in the last years may be related, in part, to the important findings on peroxisomal membrane proteins. Analysis of peroxisome-deficient mutant cells (complementation by cell fusion) and molecular biology methods (cloning of cDNA) have been applied to examine the functions and biosynthesis of peroxisomal membrane proteins in animals and humans. There is evidence that membrane proteins may play a specific role in the import machinery [211] and assembly of peroxisomes [58, 59]. Moreover, the unique localization of the D H A P - p a t h w a y enzyme activities in the peroxisomal membrane suggests an essential role for this organelle in ether lipid synthesis. Indeed, in inherited diseases with lack of normal peroxisomes, like Zellweger syndrome, patients are severely deficient in ether lipid synthesis. At the moment, it is not known whether there is a specific system for targeting enzymes, such as those of the D H A P - p a t h w a y , to the peroxisomal membrane. Except for long-chain acyl-CoA synthetases [6, 68], isolation and characterization of polypeptides (or corresponding cDNA) for these enzyme activities remain to be performed. Afterwards, studies of gene regulation may lead to knowledge of the physiological role of plasmalogens in membranes beyond what is known presently. The recent obtention of macrophage-like cell mutants defective in D H A P - A T activity but containing functional peroxisomes [149] will probably shed light on many of these questions.

Acknowledgments This work has been supported by grants from ARC, INSERM, Fondation pour la Recherche M6dicale, Ligue Nationale Franqaise contre le Cancer, and GIS Toxicologie Cellulaire of Dijon. We thank Laboratories Sterling-Winthrop and Fournier for their generous gifts of ciprofibrate and fenofibrate, respectively.

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