Peroxisome Targeting Signal of Rat Liver Acyl-Coenzyme a Oxidase

Home , Urate oxidase

MOLECULAR AND CELLULAR BIOLOGY, Jan. 1989, p. 83-91 Vol. 9, No. 1 0270-7306/89/010083-09$02.00/0 Copyright C 1989, American Society for Microbiology Peroxisome Targeting Signal of Rat Liver Acyl-Coenzyme A Oxidase Resides at the Carboxy Terminus SHOKO MIYAZAWA,l TAKASHI OSUMI,'* TAKASHI HASHIMOTO,1 KYOKO OHNO,2 SATOSHI MIURA,2 AND YUKIO FUJIKI2 Department of Biochemistry, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390,1 and Division of Molecular Cell Biology, Meiji Institute of Health Science, 540 Naruda, Odawara, Kanagawa 250,2 Japan Received 6 June 1988/Accepted 6 October 1988

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experinents with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX resides at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

The peroxisome is a subcellular organelle bounded by a When the radiolabeled proteins that were synthesized from single membrane, and it contains catalase and at least one rat liver mRNA in vitro were incubated with postnuclear H202-producing oxidase (6). Peroxisome or its relatives supernatant or highly purified peroxisomes of rat liver, some (plant glyoxysome and Trypanosoma glycosome) are present of the peroxisomal proteins were incorporated into the in almost all types of eucaryotic cells. In higher animals organelle, as deduced by the resistance to externally added including humans, this organelle plays crucial metabolic protease. Prompted by the success of the in vitro import roles, as indicated by the severe symptoms seen in Zellweger assay and with the cDNAs at hand, we investigated the syndrome and other hereditary diseases in which the biogen- peroxisome targeting signal in vitro, using various mutant esis of peroxisome is impaired (43). peroxisomal proteins. We report here evidence that rat liver Recent studies revealed that peroxisomes do not originate AOX carries the targeting information at its C terminus. from other organelles such as endoplasmic reticulum. All peroxisomal proteins are synthesized in the cytosol on free MATERIALS AND METHODS polysomes and are transported posttranslationally into the Construction of mutant AOX cDNAs. Full-length AOX preexisting organelle (for a review, see reference 23). By cDNA was constructed by combining the cDNA inserts of occasional fission, peroxisomes may form new organelles pMJ125 and pMJ131 (29). The resulting full-length cDNA and also often fuse into a larger structure called the peroxi- was inserted at the Sacd site of pTZ18R downstream of the some reticulum (24). Morphological studies (15, 16, 51, 52) T7 promoter (Fig. 1). Deletion mutants were mostly con- provide data to support this idea. structed by partial digestion with one or two appropriate Most peroxisomal proteins are synthesized at their final restriction enzymes followed by blunt ending with T4 DNA sizes. This implies that information about proteins targeted polymerase, as required, and recircularization with T4 DNA to peroxisome may reside in the mature polypeptide se- ligase. A1626 (for the designation of the mutants, see the quence. It is essential to identify this topogenic signal as a legend to Fig. 2) was constructed by deleting the sequence step to elucidate the mechanisms of peroxisome biogenesis. between two Acclll (positions 45 and 78) sites, and A226-521 Amino acid sequences (determined directly or deduced from was between the EcoRV (residue 673) and Hincll (position the gene sequences) for a number of the proteins of peroxi- 1561) sites. All other internal deletion mutants were similarly some and its related organelles have been reported (3, 7, 13, constructed. For the construction of the N-terminal deletion 18, 19, 21, 25, 27, 29-35, 37, 39-42, 47-50). Sequence mutants, the full-length plasmid was digested with AccIII, comparisons have revealed no clue as to the potential treated with nuclease BAL 31, and recircularized. One of the targeting signals of the proteins, although the importance of resulting plasmids (Al-9) lost the coding sequence up to several sequence elements for glycosomal proteins of Try- nucleotide 183 and the entire 5'-noncoding region. Al-277 was panosoma brucei and a related organism has been discussed produced from Aj69 by deleting the sequence between two (47, 50). Extensive functional analyses on various segments NcoI (positions 206 and 830) sites. A,l26 was constructed by of the protein sequences, using appropriate import assay subcloning the SspI (position 1225)-SphI (in the vector) systems, are required to clarify these topogenic signals. fragment between the SmaI and SphI sites of pTZ18R. For Recently, in vitro import assay of peroxisomal proteins these N-terminal deletion mutants, translation was expected has been achieved for several rat liver proteins, including to start at the first ATG triplet in the respective cDNA acyl-coenzyme A oxidase (AOX) and catalase (11, 20). inserts. Mutants lacking the extreme C-terminal sequence were constructed by linking the BainHI site at position 1385 * Corresponding author. (A464-660) or 1772 (&593-660) to the BamHI site of the vector. 83 84 MIYAZAWA ET AL. MOL. CELL. BIOL.

5' 3' 0- O a _.N - ~ P._ _

-t 0O -- 0- 0 CM 0 COt 0 *o b -EE a < z z z u Z Z Inma t In0 Ia. _. .. %I I .. -,.w -, mi I mow I I I I I I I I 100 200 300 400 500 600 661

C, a 0F pTZIBR FIG. 1. Restriction map of the full-length construct. Solid bar indicates the coding region of AOX cDNA. 5' (30-base-pair)- and 3' (29-base-pair)-noncoding regions are shown by a line. Restriction sites used in the construction of mutants are shown with numbers at the first positions of recognition sequences, taking the first nucleotide of the initiator methionine codon as 1. Numbers of amino acids are also shown at every 100th residue.

These mutants only retain Leu-661 at the C termini, which were verified by coupled in vitro transcription and transla- was provided by the vector sequence. For the construction tion and by immunoprecipitation with specific antibodies of M657, the plasmid containing the full-length cDNA was followed by sodium dodecyl sulfate-polyacrylamide gel elec- partially digested with PstI (only at position 1969), and then trophoresis (SDS-PAGE) and fluorography. it was blunt ended and recircularized. This treatment re- In vitro transcription and translation. The plasmids were sulted in a 4-base deletion at positions 1970 linearized by digestion with an appropriate restriction en- to 1973, thereby changing the amino acid sequence after zyme downstream of the cDNA sequences and were tran- residue 656, as described in legend to Fig. 2. For the scribed in vitro, using T7 RNA polymerase and as recom- construction of A431-661 and A658 661, a synthetic oligonucle- mended by the manufacturer (U.S. Biochemical Corp., otide linker TGTGATCACA was inserted at a PstI (position Cleveland, Ohio). In a typical experiment, 1 ,ug of plasmid 1283 or 1969) site which had been blunt ended. A464-656 and DNA was transcribed in 25 p.1 of transcription mixture, of A593-656 were constructed by inserting a double-strand linker which 1 p.1 was used for translation in a rabbit reticulocyte composed of GATCCTGCAGTCCA and AGCTTGGACTG lysate cell-free protein-synthesizing system with [35S]methi- CAG between the BamHI (position 1385 or 1772) and onine as the label (total volume, 50 ,u1). HindIII (position 1978) sites. In vitro import assay. Rat liver was homogenized in 0.25 M For the construction of fusion proteins of AOX with sucrose-10 mM Tris hydrochloride (pH 7.4)-i mM EDTA. Escherichia coli dihydrofolate reductase (DHFR) or chlor- The translation mixture was centrifuged at 8,500 x g for 10 amphenicol acetyltransferase (CAT), the dhfr and cat struc- min, and the resulting supernatant (30 ,ul) was mixed with a tural genes were inserted into pTZ19R downstream of the T7 postnuclear supernatant fraction of rat liver containing 90 p.g promoter. For DHFR fusion, the plasmid was first cleaved of peroxisomal protein (total volume, 120 p.1) as described partially with EcoRI (only at the site in the dhfr structural previously (11). The import reaction mixture also contained gene but not in the vector), blunt ended by a filling-in 0.25 M sucrose-5 mM methionine-1 mM ATP-3 mM MgCI2- reaction with the Klenow fragment of E. coli DNA polymer- 50 mM KCl. After incubation at 26°C for 60 min, the import ase I, and then digested with SacI. The full-length AOX mixture was separated into three 40-pIl aliquots. Two of plasmid was cut at one of the BamHI sites (position 1385 or these were treated with proteinase K (1/200 of the sample on 1772), filled in, and cleaved with Sacd. The resulting BamHI- the protein basis; usually ca. 200 ,ug/ml) for 30 min at 0°C, Sacd fragments were ligated to the above EcoRI-SacI-di- one in the absence and the other in the presence of 1% gested dhfr plasmid. For CAT fusion, the starting cat plas- sodium deoxycholate-1% Triton X-100. The remaining ali- mid in pTZ vector was cleaved partially with ScaI (only at quot was placed on ice for 30 min in the absence of the site in the cat structural gene) and then with KpnI. A proteinase K and detergents. As a zero-time control, another double-stranded linker composed of GGATCCAGTAAGC 40 p.1 of import mixture was digested with proteinase K TTTGAGGTAC and CTCAAAGCTTACTGGATCC was in- immediately after the translation products and postnuclear serted between these sites. The resulting plasmid was supernatant were mixed. The digestion was halted by the cleaved with BamHI and HindIII (both in the linker region) addition of 40 p.g of phenylmethylsulfonyl fluoride. Super- and ligated to the BamHI (position 1385 or 1772)-HindIII natants and peroxisome-containing pellets were separated (position 1978) fragments of AOX cDNA. The C-terminal by centrifugation, and one-fifth aliquots of both fractions mutants of the fusion proteins were constructed by replacing were analyzed by SDS-PAGE and fluorography. When nec- the AOX cDNA fragments of the fusion genes with the essary, immunoprecipitation with anti-AOX antibody was corresponding fragments of the mutant AOX cDNA. carried out as described previously (12). All the constructs were verified for nucleotide sequences Isopycnic centrifugation of import assay mixture. A light at the end points of deletions or fusion points by restriction mitochondrial fraction (300 p.g of peroxisomal protein; see mapping, sequencing, or both. The encoded polypeptides reference 5) was used for 400 p.1 of import assay. Import was VOL. 9, 1989 PEROXISOME TARGETING SIGNAL OF RAT ACYL-CoA OXIDASE 85 carried out as described above, except for the 15-min incubation in the absence of MgCl2. After treatment with proteinase K, the reaction mixture was loaded onto a 30% temperatureCC) < 261 Nycodenz (5-[N-2, 3-dihydroxypropyl acetamido]-2, 4, 6- time (mmin): r -- - 60 - triiodo-N,N'-bis[2, 3-dihydroxy-propyl]isophthalamide; Nye- proteinase K: + - + + gaard Co., Oslo, Norway) solution and centrifuged at 131,000 DOC/Triton: - - - + T So Po Si P1 S2 P2 S3 P3 x g for 60 min in a Beckman 80Ti rotor, as described Import previously (14). The peroxisomal pellet was recovered from the bottom of the gradient and analyzed by SDS-PAGE and A Full -~ _- fluorography for the imported polypeptides, for protein content (4), and for the activities of the following marker enzymes: catalase for peroxisomes (1), malate dehydrogenase B Al-277 - - ++ for mitochondria (8), esterase for microsomes (2), and N- acetylglucosaminidase for lysosomes (9). Isolation of peroxisomes. The liver of a rat that had been c A1-426 -t ++ injected with Triton WR-1339 (26) was homogenized in 0.25 M sucrose-10 mM HEPES (N-2-hydroxyethylpiperazine- 16 N'-2-ethanesulfonic acid)-KOH (pH 7.5)-i mM EDTA-0.1% D 463-59 1 -_M + dewOm A ethanol. Peroxisomes were isolated by equilibrium density centrifugation of a light mitochondrial fraction (5) in a linear E L 429-656 ) 4- JO + sucrose gradient (30 to 60%, wt/wt) in a Beckman VTi65.2 &p vertical rotor. The sucrose solution contained 50 mM glycylglycine (pH 7.5)-i mM EDTA-0.1% ethanol. Centrif- F A464-656 1.11 + ugation was carried out at 230,000 x g (average) for 90 min w at 4°C. The isolated peroxisomes were estimated to be -95% pure by means of marker enzyme assays and SDS-PAGE G A593-656 ± (10). l 1 | wz h! a RESULTS H A592-658 ...r ± Import of AOX synthesized from the cDNA by in vitro transcription and translation. To construct various mutant I A464-660 i~~~~~~~~~~~~~~~~~~~~~~~~:,- -- AOX polypeptides, we first cloned the complete AOX cDNA in a plasmid, pTZ18R, downstream of the T7 promoter (see Materials and Methods). This plasmid, encoding J A593-660 > 3 u the full-length AOX composed of 661 amino acids, gave upon in vitro transcription and translation a single polypeptide of 75 kilodaltons (Fig. 2A, lane T), which comigrated in K M657 t3 SDS-PAGE with component A of purified AOX (see below) and was immunoprecipitated with anti-AOX antibody (see FIG. 2. Posttranslational import assay of full-length and trun- Fig. 8). When the translation mixture containing this poly- cated AOX. The conditions of the import reaction and further peptide was incubated with a postnuclear supernatant frac- treatments are indicated at the top (for details, see Materials and tion of rat liver for 60 min at 26°C, the polypeptide associated Methods). DOC, Sodium deoxycholate. Lanes: T, total translation to a significant extent with the particles (Fig. 2A, lanes S, product, 1 ±l; S and P, supernatant and particle fractions, respec- and P1). Nearly half of this associated polypeptide was tively, obtained by centrifugation after the reaction experiments K indicated. Results from typical constructs are shown in panels A to resistant to externally added proteinase (Fig. 2A, lane P2). J. (A) Full-length AOX. (B and C) Polypeptides truncated at the Treatment with 1% sodium deoxycholate-1% Triton X-100 N-terminal region. (D to H) Polypeptides containing internal dele- abolished the protease resistance (Fig. 2A, lanes S3 and P3). tions in the C-terminal region. (I and J) Polypeptides with deletions When the import reaction mixture was treated with protein- in the regions encompassing the extreme C terminus. (K) Polypep- ase K without incubation at 26°C, no polypeptide was tide with a mutation in the extreme C-terminal sequence. After the recovered in the particle fraction (Fig. 2A, lanes SO and PO). import assays, labeled polypeptides were directly electrophoresed, These results indicate that some of the full-length AOX except those in panel C in which the immunoprecipitates with polypeptide was translocated posttranslationally into a mem- anti-AOX antibody were analyzed. In that case, the polypeptides brane-bound compartment in a time-dependent fashion. synthesized from the endogenous RNA closely migrated with the are to be two AOX mutant polypeptides. The AOX polypeptides synthesized from the In rat liver, there likely types of cDNAs are marked with arrowheads and an arrow. The results of polypeptides that differ in amino acid sequences between import assays are expressed at + +, +, ±, and -, as the efficiency residues 90 and 143 (29). These polypeptides are derived of import (defined as the density of the band in lane P2 relative to the from two mRNAs produced by differential splicing of the sum of those in lanes S, and P1) decreases. Deletion mutants are precursor RNA (38). No difference was found in terms of represented as Am-", where m and n specify the start and end import activity between both types of polypeptides, includ- positions of the deletion in the amino acid sequence. In M6S7. five ing all the truncated mutants tested (data not shown). C-terminal amino acid residues of AOX were mutated to -R-P-S-F- Therefore, no differentiation is made in this report for the E-V-S-L-G-H-V-COOH, with six additional residues on the C two types of AOX. terminus, compared with the authentic C-terminal sequence -L-Q- To verify that AOX was imported into the peroxisomes, S-K-L-COOH. the import reaction mixture, after treatment with proteinase K, was fractionated by Nycodenz-density gradient centrifu- 86 MIYAZAWA ET AL. MOL . CELL . BIOL .

A Full 61-277 a 429-656 -\ I / \

_ 1 2 3 4 5 proteinase K - + + SI P S2 P2 czt Pt S2 P2 S, P1 52 P2

75kDa- FIG. 4. Import of AOX polypeptides into highly purified peroxisomes. Import assays were carried out as described in Materials and Methods, except that peroxisomes (90 ,ug of protein) purified by .. were am .*43kDa sucrose-density gradient centrifugation used instead of the postnuclear supernatant. Fractions recovered from the gradient were used directly for the assay, without further treatment to remove sucrose or to concentrate the peroxisomes. Only fractions from lanes Sl, P1, S2, and P2 (for definitions, see the legend to Fig. B 2) are shown. A half aliquot was analyzed for lanes S1 and S2. The broad faint band in lane S2 of A429656 was caused by an undigestible endogenous polypeptide. Cat alase MDH Esterase NAG 4 .E 1.0 1.0 manuscript in preparation). In this system, incorporation of 3 the full-length polypeptide was also evident (Fig. 4). (12 Polypeptides deleted in the N-terminal region have high CL 0.6 0.6 10 import activities. A truncated AOX polypeptide lacking a large part of the sequence on the N-terminal side (A1l277) was 0.2 a) 0.2 imported, as well as the full-length polypeptide (Fig. 2B). A c1 0 0 time-course experiment showed that A1-277 was imported at a comparable rate with that of the full-length polypeptide FIG. 3. Isopycnic centrifugation of import assay mixture. Cen- (Fig. 5). Half-maximal incorporation (taking the amounts of was out assay K trifugation carried after import and proteinase the polypeptides incorporated after 60 min as the maximum) treatment, as described in Materials and Methods. (A) Import of the polypeptides into peroxisomes. Lanes: 1, 2, and 4, total translation was achieved in 12 and 13 min for full-length and A1-277 products without RNA, with full-length AOX mRNA, and with polypeptides, respectively. Density gradient fractionation A1-277 AOX polypeptide mRNA, respectively; 3 and 5, peroxisome confirmed that this polypeptide was also imported posttrans- fractions recovered from the Nycodenz gradient after import assay lationally into peroxisomes (Fig. 3A, lane 5), as was seen in with full-length (lane 3; 75 kilodaltons, -10 p.g of peroxisomes) and the import of full-length AOX. Furthermore, purified pero- A1-277 (lane 5; 43 kilodaltons, -15 p.g of peroxisomes) polypeptides. xisomes incorporated this polypeptide with an efficiency A major endogenous RNA translation product is indicated by the similar to that of the full-length AOX (Fig. 4). All other asterisk. (B) Activities of marker enzymes in the recovered peroxi- polypeptides, truncated in the sequences as far as up to somes. MDH, Malate dehydrogenase; NAG, N-acetylglucosamini- residue 426 in the N-terminal region A16-26, dase. Symbols: O, light mitochondrial fraction; El, isolated peroxi- (A1-69, A1426, 470-277, A32-307), exhibited comparable import ac- somes after import assay with full-length AOX; 5, those with /&157-265, A1-277- Marker enzyme activities are expressed in specific activities tivities (Fig. 6). relative to those of the light mitochondrial fraction (1.36 U/mg for catalase, 2.37 U/mg for MDH, 0.84 U/mg for esterase, and 56.7 U/mg for NAG).

0~~ ~ ~ ~ ~ ~ . gation (14) (Fig. 3A). In this experiment, we used the light mitochondrial fraction (5) instead of the postnuclear supernatant, and the reaction experiment was allowed to run for 0~~~~~~~ only 15 min. These modifications improved the separation of peroxisomes from other organelles. Peroxisomes were recovered as loose pellets at the bottom of the gradients. 2 m 2 Compared with the light mitochondrial fraction, catalase- specific activity of the peroxisome fraction increased approximately threefold, whereas other marker enzyme activ- h.6 ities decreased extensively (Fig. 3B). This indicates that the M peroxisomes were purified to a considerable extent by the 00 centrifugation. Full-length AOX cosedimented with peroxisomes (Fig. 3A, lane 3), thereby indicating that the AOX polypeptide is imported into peroxisomes. All of the above 0 15 30 45 60 results with full-length AOX are consistent with the data on Time(min) in vitro import (11) obtained using total polysomal RNA of FIG. 5. Time course of of and A1-277 AOX rat liver, in which the imported polypeptide was shown to import full-length polypeptides. The import reaction experiments were carried out as cosediment with peroxisomes but not with mitochondria on described in the legend to Fig. 2. At the indicated times, aliquots of sucrose-density gradient centrifugation. In the later part of the import mixtures were withdrawn for analysis by SDS-PAGE and this investigation of the topogenic signal of AOX, an efficient fluorography. The intensities of the bands in lanes P2 were measured in vitro import system of AOX was established by using by densitometric scanning and are expressed in arbitrary units. highly purified peroxisomes (-95% pure; S. Miura et al., Symbols: 0, full-length polypeptides; 0, A1-277 polypeptides. VOL. 9, 1989 PEROXISOME TARGETING SIGNAL OF RAT ACYL-CoA OXIDASE 87

1 200 400 600 661 Import compared with those of the full-length polypeptide and those I 1 I 1 truncated at the N-terminal region. Other constructs carry- Full 6U ++ ing internal deletions in the C-terminal side of sequence Al-69 4+ (A226-521, A464-5219 A523-591) exhibited similar levels of import Al-277 U ++ activities (Fig. 6). U Al -426 +,4, The C-terminal deletion constructs included a series of U mutants in which short stretches of amino acid residues at A16-26 * U ++ the extreme C-terminus were attached to otherwise import- A157-265 - - - N-terminal U ++ negative (see below) sequences. Polypeptides A70-277 - ++ A429_656 and A464656, which retained the five residues at the U extreme C terminus, were both imported (2 and 3%, respec- A32-307 m -+, tively; Fig. 2E and F) at levels similar to that of A463-591 On _ A226-521 - + the other hand, two other mutants in which the extreme C-terminal five or three residues were attached to another A463-591 - + N-terminal polypeptide (A593-656 and A592-658) exhibited im- A464-521 + port activities which were hardly detectable (Fig. 2G and H). A523-591 - ,+ Only after a three to four times longer fluorographic expo- A | sure than usual was a faint band seen in lane P2. Thus, the A429-656 C-terminal short amino acid sequence has the ability to make A464-656 * + the truncated N-terminal polypeptides import-positive when to A593-656 - * ±+ it is attached the C termini of such polypeptides, but this t effect is highly dependent on where it is attached. A low A592-658 - m level of incorporation into peroxisomes was also reproduced for A429-656, using purified peroxisomes (Fig. 4). A431-661 - Polypeptides deleted or mutated at the extreme C termi- A464-660 - ~ nus are not imported. Polypeptides 464- and A593-6 A593-660 - , - were fully susceptible to proteinase K digestion after incu- - bation with the postnuclear supernatant fraction, thereby A658-661 - indicating that these polypeptides were not imported (Fig. 21 A659-661. - Met - and J, lane P2). Even after a prolonged exposure, no band Met was detected in lane P2. Certain amounts of the polypeptides M657- Mm7rll - cosedimented with particulate fractions upon centrifugation 657 667 (lane P1). However, this apparent binding was not reproduc- FIG. 6. Summary of import activities o f mutant AOX polypep- ible, and we do not know whether it reflected specific tides. Solid bars indicate the regions co)vered by the respective binding; it could be due to aggregation of the polypeptides polypeptides. For a definition of import acttivities, see the legend to during incubation. A similar construct lacking the C-terminal Fig. 2. The triangle indicates the cleava ge site of the AOX A sequence (A431-661) was also import negative (Fig. 6). To test component to yield the B and C componerits (see text). the importance of the extreme C terminal sequence, we constructed a plasmid which specifies a different amino acid sequence after residue 656 (M657). Four of five amino acids In a typical experiment, the follox%ving values of import were mutated, and six extra residues were added at the activities (percent incorporation during 60 min) were ob- terminus. This mutant polypeptide was completely incapable tained by densitometric scanning of the fluorograms: full- of import (Fig. 2K). Two other mutants, one with a deletion length polypeptide, 16%; A1-277, 22%; and A1l26, 15%. For of the C-terminal four residues (A658,661) and the other with A11426, two translation products (one nnajor and minor) were a deletion of the C-terminal three residues but the addition of detected (Fig. 2C, arrowhead and arrc w). In this construct, a methionine residue (A659661 Met), were also import nega- translation may start at Met-436 as we 11 as Met-427, the first tive (Fig. 6). ATG triplet in the truncated cDNA se quence (cf. reference C-terminal sequences of AOX exhibit peroxisome target- 29). The relative amounts of the twlo polypeptides were ing activities when fused to foreign proteins. To determine reversed in the particle fraction aftter the import assay whether the C-terminal region of AOX has sufficient infor- (compare lanes Sl, P1, and P2 in Fig. 2 ). This result might be mation to direct proteins to peroxisomes, we carried out due to a different import efficiency ofthie two polypeptides or fusion experiments. Two E. coli enzymes, DHFR and CAT, to an intraperoxisomal processing rezaction occurring near were chosen as fusion partners because they were likely to Met-436, although the correct cleavage site is located further lack any intrinsic organelle targeting information. downstream by -30 amino acid residu es (see below). Some When the C-terminal 199 acid residues of AOX were fused of the amino-terminal deletion mutanLts were translated at to DHFR near the C terminus, the fusion protein was lower efficiencies than the full-length i kOX (e.g., A1 26; see imported (Fig. 7A, DHFR-AOX199). When the extreme Fig. 2C) and thus were used to lower concentrations in the AOX C-terminal sequence of the fusion protein was changed import assays. This was not, howevei r, the reason for their as in M657, import no longer occurred (Fig. 7B, DHFR- apparent high import efficiencies, becaiuse even when 1/10 of AOX199M657). A fusion protein of DHFR with the C- the full-length AOX (as in Fig. 2A) wais used, the efficiency terminal 70 residues of AOX (DHFR-AOX70) gave an am- of import (expressed as percent incorporation) remained biguous result. A very faint band appeared in lane P2 only unchanged (data not shown). after prolonged fluorographic exposure. This band was ab- Internal deletions in the C-termir ial region lower the sent in the corresponding C-terminal M657 mutant (data not import activities. Polypeptide A463591 exhibited a much less shown). efficient but still significant import a(ctivity (2%; Fig. 2D) Fusion experiments were also carried out with CAT. 88 MIYAZAWA ET AL. MOL. CELL. BIOL.

temDerature( C) -26 -> Lime (min): - .60 -- proteinase K: - - - DOC/Triton :

T So PR S. P' S2 Pz- 3 P3 Import 140 463 661 A DHFR-AOX 1a9 + 140 463 656 667 B DHFR-AOX 1 99M65-7 212 463 661 C CAT-AOX 199 + liBii.-t 212 463 656 6567 D CAT-AOX199M65s7 212 592 661i E CAT-AOX70 +

212 592656 667 F CAT-AOX70M657

FIG. 7. Import of fusion proteins. E. coli DHFR consists of 159 amino acids. In DHFR-AOX199, DHFR polypeptide was truncated at position 140 and the C-terminal AOX polypeptide of 199 residues was attached. In DHFR-AOX199M657, the extreme C terminus of the AOX moiety was modified as in M657 (see the legend to Fig. 2). Authentic CAT contains 219 amino acids. In CAT-AOX199 and CAT-AOX70, the C-terminal AOX polypeptides of 199 and 70 residues were linked to CAT at position 213. CAT-AOX199M657 and CAT-AOX70M657 have the M657 mutation at the extreme C termini. Open bars indicate DHFR and CAT sequences; filled bars denote AOX sequences. Hatched areas indicate the modified sequences at the extreme C termini. Residues are numbered using the sequence position numbers of the respective authentic proteins. Import assays were carried out as described in the legend to Fig. 2. Bands of the respective polypeptides are marked with arrowheads. Import activities as defined in the legend to Fig. 2 are shown on the right.

When the C-terminal 199 residues of AOX were fused to bands corresponding to component B were also found in CAT, the resulting protein was import positive (Fig. 7C, samples not treated with the protease (lanes P1). In the case CAT-AOX199). When the C terminus of the AOX part of the of the full-length polypeptide, bands were seen in lane S2 fusion protein was mutated to the M657 sequence, the close to the positions of components B and C (vertical resulting mutant polypeptide was not imported (Fig. 7D). A arrows). These were likely to be formed by the cleavage of fusion protein of CAT with the C-terminal 70 residues of some of the full-length polypeptides by proteinase K be- AOX (CAT-AOX70) was also imported, although with a tween the two domains (see Discussion) near the authentic much lower efficiency than that of CAT-AOX199 (Fig. 7E processing site. The present results clearly show that the and F). proteolytic processing of AOX is an intraperoxisomal event. We also constructed a fusion protein between CAT and The difference in the efficiency of the processing among the the last five amino acids of AOX; however, this fusion four polypeptides is probably due to differences in the polypeptide was not digested by proteinase K. Thus, it was conformations caused by the deletions. not clear whether the polypeptide was imported. Complete digestion of the unincorporated fusion polypeptide with DISCUSSION higher amounts of proteinase K, even up to 10 times as much as used in the usual assays, as well as with several other The present results are summarized as follows. (i) Dele- proteases (e.g. thermolysin), was unsuccessful. Both DHFR tions in the N-terminal region of AOX have no apparent and CAT were also highly protease resistant. effect on the import activity. (ii) All internal deletions Intraperoxisomal processing activity. We suggested in a encompassing the C-terminal region reduce the import ac- previous report (36) that purified AOX contains three poly- tivity. (iii) A deletion (e.g., A658-661 and A659 661. Met) or peptide components, A, B, and C, the latter two being mutation (i.e., M657) involving the extreme C terminus formed by endoproteolytic cleavage of component A. A causes a complete loss of import activity. (iv) The C-terminal protein-labeling experiment using isolated hepatocytes (28) 199 and 70 amino acid stretches of AOX contain sufficient verified this processing and further showed that the cleavage peroxisome targeting information as was shown by the reaction occurs in vivo, most likely in peroxisomes. We fusion experiments with two bacterial enzymes, DHFR and mapped the cleavage site between Val-468 and Ala-469 CAT. When the extreme C-terminal sequence is mutated, (shown by the triangle in Fig. 6), locating the B and C these stretches are unable to direct the fusion proteins to components (53 and 22 kilodaltons, respectively) on the N- peroxisomes. (v) The C-terminal sequence of five amino and C-terminal sides, respectively (29). acids (residues 657 to 661) rescues translocation abilities In the present study, we detected this processing activity when linked to otherwise import-negative polypeptide moi- in vitro (Fig. 8). The full-length polypeptide and some of the eties of AOX. The sequence 657 to 661 is clearly effective on N-terminal deletion mutants (A169, A16-26, and A32-307) gave two different amino acid stretches of AOX, i.e., residues 1 to bands corresponding to the respective component B (smaller 428 and 1 to 463. This finding suggests that the rescue of in the case of the mutants than the authentic component B import activities is not simply due to the combination of the because of deletions) in the particle fractions (lanes P1 and C-terminal amino acid residues and sequences near the P2) but not in the supernatant fractions (lanes S1). Compo- junctions. nent C was hardly detectable because of its low methionine The most consistent interpretation of the present data is content. This proteolytic cleavage is not caused by an that the targeting signal of AOX resides at the C terminus of artifact produced by proteinase K treatment because the the enzyme. Within this region, five or fewer amino acids VOL. 9, 1989 PEROXISOME TARGETING SIGNAL OF RAT ACYL-CoA OXIDASE 89

Full A61-69 A 1 6-26 A 32-307

Proteinase K: - + + - - + - + T Si Pi S2 P2 T Si Pi S2 P2 T Si Pi82 P2 T Si Pi S2 P2 200-

97- 68- A _ m4 .. low _.' -4 '- 4 o_w 4.... 45 -4 g' a3m - X 4040-M -, 4 26- 18- (kDa) 4 Processing (%): 15 51 24 20 FIG. 8. Intraperoxisomal processing of AOX. Full-length and three N-terminal deletion mutant polypeptides after the import reaction were immunoprecipitated with anti-AOX antibody and analyzed by SDS-PAGE. For the designation of the lanes and the constructs, see the legend to Fig. 2. Positions of size markers (in kilodaltons) and three components (A, B, and C) of purified AOX are shown at the left. Filled and open triangles denote the bands corresponding to the A and B components, respectively, for each construct. The two vertical arrows in lane S2 of full-length AOX indicate the cleavage products of AOX by proteinase K (see text). The efficiencies of processing are expressed as percent conversion (B/A+B) calculated based on the relative densities (measured by densitometric scanning) of the bands in lanes P2, taking into account the methionine contents of the respective components.

at the extreme C terminus are essential for import (cf., (A46656 - A429-656 >> A593 656; Fig. 2), which depends on A658-661, A659-661, Met and M657). The results summarized in where the C-terminal five residues are attached. Residue 464 items iv and v suggest that the targeting function is localized is located almost exactly at the boundary of domains B and in this short sequence. Because of technical difficulties, we C, thus the C-terminal residues attached here would be did not demonstrate by a fusion experiment that this se- rather free from the conformation of the rest of the protein quence satisfied the minimal requirement for import (see body. Residue 429 is in the end region of domain B, whereas Results). Hence, we cannot exclude the remote possibility residue 593 is at the middle of domain C. Thus, the C- that the N-terminal stretch of AOX up to residue 428 (though terminal residues placed at these three different positions neither necessary nor sufficient by itselffor import) has some would be subjected to increasing structural constraints. The structure that acts as a targeting signal only by cooperating effect of conformation might also explain the different func- with the C-terminal short sequence. tional activities of the C-terminal sequences used in the It is clear from the results summarized in item ii that the fusion experiments. That is, the 199-residue sequence, internal sequence on the C-terminal side (residues 429 to which contains the entire region of domain C and thus 656, although the exact boundaries are yet to be determined) maintains the C-terminal short sequence in the most proper also plays an important role in the efficient import of the conformation, confers a rather strong signal function. On the enzyme. We previously suggested (29) that AOX is composed of two structurally independent domains which can be separated by intraperoxisomal proteolytic processing into mature components B and C, as described above. In the present work, we reproduced this processing in a cell-free system (Fig. 8). As shown in Fig. 9, most of domain B has no apparent function in targeting. On the other hand, the C-terminal internal region that has an enhancing effect on import corresponds approximately to domain C, thereby suggesting the functional importance of this domain to import. Several explanations can be given for the function of domain C. (i) A secondary signal which functions coopera- NH2 Targeting tively with the C-terminal signal is included in this domain. signal However, this sequence does not work independently. (ii) Some structure in domain C supplies the extreme C-terminal I II I signal with a proper conformation to be recognized or I It exposes it free on the molecular surface. (iii) Domain C is FIG. 9. Proposed domain structure of AOX. Region I (residues 1 required not for targeting itself but for efficient retention of to 426) has no apparent function in targeting, whereas region II the polypeptide in peroxisomes. (residues 429 to 656) plays an important role in the efficient import We favor the second process. The boundary of these regions was tentatively determined. possibility. The targeting activity of The processing site is between residues 468 and 469. The targeting the extreme C-terminal seems to be strongly affected by the signal is located in the C-terminal stretch of amino acids, including protein conformation. This fact is inferred by the gradation the five or fewer residues at the extreme terminus. The locations of of import efficiencies of three different AOX polypeptides domains B and C are shown. 90 MIYAZAWA ET AL. MOL. CELL. BIOL.

AOX |K H L K P tion, although they do possess a significant homology of -QI overall sequence (29). Bifunctional enzyme S L A G EH G V/ L/7 'S'K'L ACKNOWLEDGMENTS Luciferase NA K[G G K S//L We thank I. Kasuya for expert technical assistance and M. Ohara Uricase 11 A SgS RWIW for critical comments. We thank M. Iwakura and B. Howard for providing the plasmids containing dhfr (pTP64-1) and cat (pSV2cat) Malate synthase I EH P R E L genes, respectively. D-Amino acid oxidase N LWIT M P P This work was supported in part by Grants-in-Aid for Scientific EIU Research from the Ministry of Education, Science, and Culture of FIG. 10. C-terminal sequences of AOX and other peroxisomal Japan. proteins. Sequences were taken from the references cited in the LITERATURE CITED text. Amino acids are represented by standard one-letter codes. Residues are boxed when identical between AOX and other proteins 1. Baudhuin, P., H. Beaufay, Y. Rahman-Li, 0. Z. Sellinger, R. at the corresponding positions. The three hatched amino acid Wattiaux, P. Jacques, and C. de Duve. 1964. Tissue fractionation residues are common sequences among the five peroxisomal pro- studies. 17. Intracellular distribution of monoamine oxidase, teins. aspartate aminotransferase, alanine aminotransferase, D-amino acid oxidase and catalase in rat-liver tissue. Biochem. J. 92:179- 184. 2. Beaufay, H., A. Amar-Costesec, E. Feytmans, D. Thines-Sem- other hand, the 70-residue sequence, which lacks the com- poux, M. Wibo, M. Robbi, and J. Berthet. 1974. Analytical study plete domain structure, has a much lower targeting activity. of microsomes and isolated intracellular membranes from rat Recently, Gould et al. (17) reported the peroxisome- liver. I. Biochemical methods. J. Cell Biol. 61:188-200. targeting signal of firefly luciferase (naturally occurring in the 3. Bethards, L. A., R. W. Skadsen, and J. G. Scandalios. 1987. peroxisomelike organelle of the lantern organ [22]). They Isolation and characterization of a cDNA clone for the cat2 gene introduced into mammalian cells various DNA constructs in maize and its homology with other catalases. Proc. Natl. Acad. Sci. USA 84:6830-6834. coding for mutant luciferase polypeptides as well as fusion 4. Bradford, M. 1976. A rapid and sensitive method for the polypeptides with certain cytosolic proteins. By examining quantitation of microgram quantities of protein utilizing the the intracellular localization of the expressed polypeptides, principle of dye-binding. Anal. Biochem. 72:248-254. they confined the targeting activity to no more than 12 amino 5. de Duve, C., B. C. Pressman, R. Gianetto, R. Wattiaux, and F. acid residues at the C terminus. Appelmans. 1955. Tissue fractionation studies. 6. Intracellular It is noteworthy that rat liver AOX (29) and firefly lucif- distribution patterns of enzymes in rat-liver tissue. Biochem. J. erase (7) share the C-terminal sequence of Ser-Lys-Leu (Fig. 60:604-617. 10). Among the ca. 20 peroxisomal proteins so far se- 6. de Duve, C., and P. Baudhuin. 1966. Peroxisomes (microbodies quenced, rat liver bifunctional enzyme (37), soybean uricase and related particles). Physiol. Rev. 46:323-357. 7. de Wet, J. R., K. V. Wood, M. DeLuca, D. R. Helinski, and S. II (31), and cucumber malate synthase (only a partial se- Subramani. 1987. Firefly luciferase gene: structure and expres- quence reported) (46) also have this sequence at the C sion in mammalian cells. Mol. Cell. Biol. 7:725-737. termini. The frequency, five in 20 proteins, is clearly much 8. Dupourque, D., and E. Kun. 1%9. Cytoplasmic and mitochon- higher than the expected frequency of appearance of a given drial malate dehydrogenases from beef kidney. Methods En- sequence of three amino acids, which is one in 8,000. In zymol. 13:116-122. addition, the C-terminal sequence of pig kidney D-amino 9. Findlay, J., G. A. Levvy, and C. A. Marsh. 1958. Inhibition of acid oxidase is Ser-His-Leu (40), with some similarity to the glycosidases by aldonolactones of corresponding configura- above five. We suspect that, in some if not all of these tions. 2. Inhibitors of ,B-N-acetylglucosaminidase. Biochem. J. 69:467-476. proteins, the short C-terminal sequence of three amino acid 10. Fujiki, Y., S. Fowler, H. Shio, A. L. Hubbard, and P. B. residues has a crucial function in the targeting process. Lazarow. 1982. Polypeptide and phosopholipid composition of The glycosomal phosphoglycerate kinase of a Kineto- the membrane of rat liver peroxisomes: comparison with endo- plastida, Crithidiafasciculata, has been proposed to contain plasmic reticulum and mitochondrial membranes. J. Cell Biol. the organelle-targeting signal at the C terminus (47). This 93:103-110. argument is based on the observation that the glycosomal 11. Fujiki, Y., and P. B. Lazarow. 1985. Post-translational import of form of the enzyme has a C-terminal extension compared fatty acyl-CoA oxidase and catalase into peroxisomes of rat with its cytosolic counterpart, whereas in other regions, the liver in vitro. J. Biol. Chem. 260:5603-5609. two isozymes have virtually identical sequences. The same 12. Fujiki, Y., R. A. Rachubinski, and P. B. Lazarow. 1984. Syn- thesis of a major integral enzyme of the T. brucei glycosome also has a C-terminal membrane polypeptide of rat liver peroxisomes on free polysomes. Proc. Natl. Acad. Sci. USA 81: extension which is not found in its cytosolic counterpart 7127-7131. (48). It mnight be worth noting that these enzymes lack the 13. Furuta, S., H. Hayashi, M. HiJikata, S. Miyazawa, T. Osumi, Ser-Lys-Leu sequence at their extreme C termini. and T. Hashimoto. 1986. Complete nucleotide sequence of Most recently, the in vitro import of AOX (PXP4 protein) cDNA and deduced amino acid sequence of rat liver catalase. of the yeast, Candida tropicalis, has been reported (44, 45). Proc. Natl. Acad. Sci. USA 83:313-317. With the in vitro import assay, it has been shown that this 14. Ghosh, M. K., and A. K. Hajra. 1986. A rapid method for the protein contains, in two separate regions, peroxisome tar- isolation of peroxisomes from rat liver. Anal. Biochem. 159: geting sequences. These sequences can act independently in 169-174. targeting the truncated polypeptides of the oxidase and the 15. Gorgas, K. 1984. Peroxisomes in sebaceous glands. V. Complex peroxisomes in the mouse preputial gland: serial sectioning and fusion polypeptides with mouse DHFR. The C-terminal three-dimensional reconstruction studies. Anat. Embryol. 169: sequence apparently has no function in this process. These 261-270. results differ completely from those for rat liver AOX given 16. Gorgas, K. 1985. Serial section analysis of mouse hepatic in the present report. Thus, AOXs of the two organisms peroxisomes. Anat. Embryol. 172:21-32. might have highly deviated peroxisome targeting informa- 17. Gould, S. J., G.-A. Keller, and S. Subramani. 1987. Identifica- VOL. 9, 1989 PEROXISOME TARGETING SIGNAL OF RAT ACYL-CoA OXIDASE 91

tion of a peroxisomal targeting signal at the carboxy terminus of Veeneman, J. H. van Boom, P. A. M. Michels, and F. R. firefly luciferase. J. Cell Biol. 105:2923-2931. Opperdoes. 1985. Topogenesis of microbody enzymes: a se- 18. Hijikata, M., N. Ishii, H. Kagamiyama, T. Osumi, and T. quence comparison of the genes for the glycosomal (microbody) Hashimoto. 1987. Structural analysis of cDNA for rat peroxi- and cytosolic phosphoglycerate kinases of Trypanosoma brucei. somal 3-ketoacyl-CoA thiolase. J. Biol. Chem. 262:8151-8158. EMBO J. 4:3811-3817. 19. Hill, D. E., R. Bowlay, and D. Rogers. 1988. Complete nucleo- 36. Osumi, T., T. Hashimoto, and N. Ui. 1980. Purification and tide sequence of the peroxisomal acyl-CoA oxidase from the properties of acyl-CoA oxidase from rat liver. J. Biochem. 87: alkane-utilizing yeast Candida maltosa. Nucleic Acids Res. 16: 1735-1746. 365-366. 37. Osumi, T., N. Ishii, M. Hijikata, K. Kamnio, H. Ozasa, S. 20. Imanaka, T., G. M. Small, and P. B. Lazarow. 1987. Translo- Furuta, S. Miyazawa, K. Kondo, K. Inoue, H. Kagamiyama, and cation of acyl-CoA oxidase into peroxisomes requires ATP T. Hashimoto. 1985. Molecular cloning and nucleotide sequence hydrolysis but not a membrane potential. J. Cell Biol. 105:2915- of the cDNA for rat peroxisomal enoyl-CoA hydratase: 3- 2922. hydroxyacyl-CoA dehydrogenase bifunctional enzyme. J. Biol. 21. Janowicz, Z. A., M. R. Eckart, C. Drewke, R. 0. Roggenkamp, Chem. 260:8905-8910. and C. P. Hollenberg. 1985. Cloning and characterization of the 38. Osumi, T., N. Ishii, S. Miyazawa, and T. Hashimoto. 1987. DAS gene encoding the major methanol assimilatory enzyme Isolation and characterization of the rat acyl-CoA oxidase gene. from the methylotrophic yeast Hansenula polymorpha. Nucleic J. Biol. Chem. 262:8138-8143. Acids Res. 13:3043-3062. 39. Quan, F., R. G. Korneluk, M. B. Tropak, and R. A. Gravel. 22. Keller, G.-A., S. Gould, M. DeLuca, and S. Subramani. 1987. 1986. Isolation and characterization of the human catalase gene. Firefly luciferase is targeted to peroxisomes in mammalian cells. Nucleic Acids Res. 14:5321-5335. Proc. Natl. Acad. Sci. USA 84:3264-3268. 40. Ronchi, S., L. Minchiotti, M. Galliano, B. Curti, R. P. Swenson, 23. Lazarow, P. B., and Y. Fujiki. 1985. Biogenesis of peroxisomes. C. H. Williams, Jr., and V. Massey. 1982. The primary structure Annu. Rev. Cell Biol. 1:489-530. of D-amino acid oxidase from pig kidney. IL. Isolation and 24. Lazarow, P. B., M. Robbi, Y. Fujiki, and L. Wong. 1982. sequence of overlap peptides and the complete sequence. J. Biogenesis of peroxisomal proteins in vivo and in vitro. Ann. Biol. Chem. 257:8824-8834. N.Y. Acad. Sci. 386:285-300. 41. Sakajo, S., K. Nakamura, and T. Asahi. 1987. Molecular cloning 25. Ledeboer, A. M., L. Edens, J. Maat, C. Visser, J. W. Bos, and and nucleotide sequence of full-length cDNA for sweet potato C. T. Verrips. 1985. Molecular cloning and characterization of a catalase mRNA. Eur. J. Biochem. 165:437-442. gene coding for methanol oxidase in Hansenula polymorpha. 42. Schroeder, W. A., J. R. Shelton, J. B. Shelton, B. Robberson, G. Nucleic Acids Res. 13:3063-3082. Apell, R. S. Fang, and J. Bonaventura. 1982. The complete 26. Leighton, F., B. Poole, H. Beaufay, P. Baudhuin, J. W. Coffey, S. amino acid sequence of bovine liver catalase and the partial Fowler, and C. de Duve. 1968. The large-scale separation of sequence of bovine erythrocyte catalase. Arch. Biochem. peroxisomes, mitochondria, and lysosomes from the livers of Biophys. 214:397-421. rats injected with Triton WR-1339. Improved isolation proce- 43. Schutgens, R. B. H., H. S. A. Heymans, R. J. A. Wanders, H. dures, automated analysis, biochemical and morphological van den Bosch, and J. M. Tager. 1986. Peroxisomal disorders: a properties of fractions. J. Cell Biol. 37:482-513. newly recognised group of genetic diseases. Eur. J. 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Furuta, H. synthase of cucumber: molecular cloning of a cDNA and Kagamiyama, T. Osumi, and T. Hashimoto. 1987. Complete regulation of enzyme synthesis during germination. Plant Phys- nucleotide sequence of cDNA and predicted amino acid se- iol. 81:762-767. quence of rat acyl-CoA oxidase. J. Biol. Chem. 262:8131-8137. 47. Swinkels, B. W., R. Evans, and P. Borst. 1988. The topogenic 30. Murray, W. W., and R. A. Rachubinski. 1987. The primary signal of the glycosomal (microbody) phosphoglycerate kinase structure of a peroxisomal fatty acyl-CoA oxidase from the of Crithidia fasciculata reside in a carboxy-terminal extension. yeast Candida tropicalis pK233. Gene 51:119-128. EMBO J. 7:1159-1165. 31. Nguyen, T., M. Zelechowska, V. Foster, H. Bergmann, and 48. Swinkels, B. W., W. C. Gibson, K. A. Osinga, R. Kramer, G. H. D. P. S. Verma. 1985. Primary structure of the soybean nodulin- Veeneman, J. H. van Boom, and P. Borst. 1986. 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Okazaki, K., T. Takechi, N. Kambara, S. Fukui, I. Kubota, and soma brucei may serve as topogenic signals for import into T. Kamiryo. 1986. Two acyl-coenzyme A oxidases in peroxi- glycosomes. EMBO J. 6:215-221. somes of the yeast Candida tropicalis: primary structures 51. Yamamoto, K., and H. D. Fahimi. 1987. Three-dimensional deduced from genomic DNA sequence. Proc. Natl. Acad. Sci. reconstruction of a peroxisomal reticulum in regenerating rat USA 83:1232-1236. liver: evidence of interconnections between heterogeneous seg- 34. Okazaki, K., H. Tan, S. Fukui, I. Kubota, and T. Kamiryo. 1987. ments. J. Cell Biol. 105:713-722. Peroxisomal acyl-coenzyme A oxidase multigene family of the 52. Yamamoto, K., and H. D. Fahimi. 1987. Biogenesis of peroxi- yeast Candida tropicalis; nucleotide sequence of a third gene somes in regenerating rat liver. I. Sequential changes of catalase and its protein product. Gene 58:37-44. and urate oxidase detected by ultrastructural cytochemistry. 35. Osinga, K. A., B. W. Swinkels, W. C. Gibson, P. Borst, G. H. Eur. J. Cell Biol. 43:293-300.