Cloning, Sequencing, and Expression of a cDNA Encoding Rat ...Printed in U.S.A. Cloning, Sequencing,...

THE JOURNAL OF BIOLOC~CAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 1.8, Issue of June 25, pp. 10720-10725,199O Printed in U.S.A.

Cloning, Sequencing, and Expression of a cDNA Encoding Rat Liver Mitochondrial Carnitine Palmitoyltransferase II*

(Received for publication, January 12, 1990)

Keith F. WoeltjeQ, Victoria Essert, Brian C. Wei&, Anjan SenS, William F. Cox$, Michael J. McPhaulS, Clive A. Slaughtergll, Daniel W. Foster+, and J. Denis McGarry$$II From the Departments of tlnternal Medicine and CBiochemistry, the IlHoward Hughes Medical Institute and the Center for Diabetes Research, University of Texas Southwestern Medical Center, Southwestern Medical School, Dallas, Texas 75235

We report the isolation and characterization of a full- length cDNA encoding rat liver carnitine palmitoyltransferase II (CPT II). Beginning with the purified protein CNBr fragments were generated and sequenced. Corresponding oligonucleotides were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone ultimately obtained consisted of a 62 nucleotide 5’-untranslated region, a single open reading frame of 1,974 bases predicting a protein of 658 amino acids (Mr = 74,119), and a 3’-untranslated segment of 260 nucleotides followed by the poly (A) tail.

The identity of the cDNA was confirmed by the findings that (a) the open reading frame encoded all three peptides found in the original protein; (b) a fourth peptide synthesized from a portion of the deduced amino acid sequence and used to immunize a rabbit resulted in the generation of an antibody that recognized pure CPT II on a Western blot; (c) in vitro transcription and translation of the cDNA (ligated into pBlue-script KS (+)) generated a protein that was specifically immunoprecipitated by anti-CPT II antibody and having a M, slightly greater than that of mature CPT II; (d) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV4, resulted in a 6-fold induction of mitochondrial CPT II catalytic activity.

It seems likely that the de nouo synthesized enzyme gains entry into the mitochondrion via a targeting peptide that is subsequently cleaved. The mature protein probably associates (relatively loosely) with the inner membrane through a limited number of membrane spanning domains.

The predicted amino acid sequence of CPT II shows strong identity with those of two other acyltransferases, namely, rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase.

Studies described in the preceding paper (1) dealt with a number of physical and functional characteristics of the mi-

* This work was supported by Grant DK18573 from the National Institutes of Health, Robert A. Welch Foundation Grant I-1090, and March of Dimes Grant 5-694. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505470.

11 To whom correspondence should be addressed.

tochondrial carnitine palmitoyltransferase (CPT)’ enzymes in several tissues and species. The information gained was important to a proper understanding of operational interac- tions in this complex fatty acid transport mechanism. Our next goal is to determine the structures of the proteins in- volved and their topographical relationship with the mitochondrial membranes. Since we had already purified rat liver CPT II and raised an antibody against it (2) our first effort was to isolate and sequence its corresponding cDNA. As outlined below, these experiments have yielded the first complete amino acid sequence of a mitochondrial CPT protein.

EXPERIMENTAL PROCEDURES

Animals-Male Sprague-Dawley rats (-150 g) maintained on a regular chow diet were used in the fed state. In some cases di-(Z- ethylhexyl)phthalate was added to the diet at 0.2% for 2 weeks prior to killing.

General Methods-Standard molecular biological techniques were employed (3). cDNA clones were sequenced on both strands using the dideoxy chain-termination method (4) with either the Ml3 universal sequencing primer or specific internal oligonucleotide primers after subcloning into bacteriophage Ml3 vectors. Sequencing reactions were performed with the Klenow fragment of Escherichia coli DNA polymerase I (4) or a modified bacteriophage T7 DNA polymerase (Sequenase) (5). Total cellular RNA was isolated bv guanidinium thiocyanate extraction followed by centrifugation &rough a CsCl cushion (6).

Protein Purification and Peptide Sequencing-Rat liver CPT II, purified as described previously (2), was digested with cyanogen bromide in 70% formic acid for 15 h in the dark and lvoahilized. The

” _

resulting peptide mixture was subjected to reverse-phase high pres- sure liquid chromatography using-a system from the Waters Chro- matopraDhv Division of Milliaore (Milford. MA), eauiDDed with an Appl&d Bibsystems Inc. (Sanka Clara, CA) kP300 c&&n. Chroma- tography was performed in 0.02% (v/v) trifluoroacetic acid with a gradient of O-50% acetonitrile. Amino acid sequencing was carried out on three of the peptides using an Applied Biosystems Inc. (Foster City, CA) model 470A automated sequenator equipped with a model 120A PTH amino acid analyzer according to the manufacturer’s standard programming and chemicals.

Screening of a cDNA Library-The 3-6-kilobase fraction of a rat liver cDNA library in the plasmid cloning vector pcDV (7) was used. It was screened by colony hybridization (3) using two 32P-5’ end- labeled oligonucleotides deduced from the amino acid sequence obtained from two of the isolated CPT II peptides (see below).

Construction of a Primer-extended cDNA Library-Poly(A)’ RNA from livers of rats treated with di-(2-ethylhexyl)phthalate (a known inducer of CPT II svnthesis (8) ) was used to construct a primer- extended cDNA library, employing a kit from Promega and a synthesized nrimer with the sequence 5’-TTGGGTCCGGATTGAAT-3’ (complementary to bases iO8-424 in Fig. 3). The double-stranded cDNA generated was ligated into hgtl0 arms (Promega) using EcoRI linkers. The ligation mixture was packaged with a two extract system

’ The abbreviations used are: CPT, carnitine palmitoyltransferase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electropho- resis; CoA, coenzyme A.

10720

by guest on March 20, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

cDNA for Carnitine Palmitoyltransferase II 10721

(Gigapack Gold, Stratagene) and the resulting library plated on a lawn of host strain E. coli C600 /$A. lo6 plaques were screened using a “‘P-hexamer-primed PstI-XhoI fragment (261 base pairs), 5’ to the internal oligonucleotide, as a probe.

(Madison, WI), and U. S. Biochemical (Cleveland, OH). “S- and “P- labeled nucleotides were from Amersham Corp. and Du Pont-New England Nuclear.

Plasmid Construction (see Fig. I)-Plasmid pBKS-CPT II.3 was constructed by removing the cDNA insert from clone pCD-CPT II.1 using StuI and NsiI and ligating this fragment into EcoRV, PstI cut pBluescriptKS(+). Plasmid pBKS-CPT II.2 was constructed by in- serting the EcoRI fragment of XL4-CPT II.1 into EcoRI cut pBluescriptKS(+). A full-length cDNA insert was then constructed in plasmid pBKS-CPT II.4 by removing the XhoI fragment of pBKS- CPT II.3 and replacing it with the XhoI fragment of pBKS-CPT 11.2.

RESULTS

For transfection experiments the SmaI fragment from pBKS-CPT II.4 was ligated into the SmaI site of the expression vector, pCMV4 (9), to yield the construct designated pCMV4CPT 11.4.

In Vitro Transcription and Translation-i-Methyl guanosine- capped RNA was made using the Stratagene capping system. Tran- script was generated from X&I linearized plasmids pBKS-CPT II.3 and pBKS-CPT II.4 (Fig. 1) using the T3 RNA polymerase. One to 2 pg of in vitro transcribed mRNA was translated in a rabbit reticu- locyte lysate system containing [?+]methionine (Du Pont-New Eng- land Nuclear). The translation product was brought to 1 ml with precipitation buffer (10 mM Tris-HCl, pH 7.4,0.1% SDS, 0.1% Triton X-100, and 2 mM EDTA) in a microcentrifuge tube. Twenty Kg of preimmune antibody RK39 or immune anti-CPT II antibody RK40(2) was added and allowed to incubate for 1 h at room temperature with rocking. Immune complexes were precipitated following addition of 50 ~1 of Pansorbin (10% suspension of killed Stuphylococcus uureus (Calbiochem) prewashed three times in precipitation buffer), rocking for 1 h, and centrifugation. The supernatant was discarded, and the pellet was washed three times with precipitation buffer. The final pellet was resuspended in 100 ~1 of SDS sample buffer (10) and boiled for 4 min. The Pansorbin was sedimented and the supernatant removed for SDS-PAGE. Eight % polyacrylamide gels were run and fixed in En”Hance (Du Pont-New England Nuclear) as described (10). The immunoprecipitated products were visualized by fluorography.

Molecular Cloning of a Rat Liver CPT II cDNA-Purified rat liver CPT II was subjected to N-terminal amino acid sequencing without success, suggesting that the amino terminus of the protein was blocked. Three cyanogen bromide cleavage peptides were, however, successfully sequenced (underlined in Fig. 3). With this information two degenerate oligonucleotide probes were synthesized, one to a region within

peptide 1 (5’-NNtTCzTGtTAtTGCAT-3’), the other to a

DNA Trunsfection-Stock cultures of simian COS-M6 cells were grown in monolayer at 37 “C under a 5% CO, atmosphere in Dulbec- co’s modified Eagle’s medium supplemented with 10% fetal calf serum, 44 mM NaHC03, pH 7.4, 100 units/ml penicillin, and 100 pg/ ml streptomycin. Cells were seeded on day 0 at a density of 3 x 106/ 150.mm dish in the above medium. On day 1, the medium was removed, and the cells were washed with buffer A (140 mM NaCl, 3 mM KCl, 0.5 mM MgCl,, 1 mM CaC12, 0.9 mM sodium phosphate, and 25 mM Tris-HCl, pH 7.4). The cells were then exposed to 15 pg of plasmid DNA in 7.5 ml of buffer A containing 0.5 mg/ml DEAE- dextran (Pharmacia LKB Biotechnology Inc., M, = 5 x 10’) (11). After 30 min at 37 “C, the DNA-containing solution was aspirated, and the cells were incubated for 3 h at 37 “C in 15 ml of fresh medium containing 100 pM chloroquine. The cells were washed with buffer A and then incubated for 4 min at room temperature with 5 ml of supplemented Dulbecco’s modified Eagle’s medium containing 20% (v/v) glycerol. After the glycerol shock, cells were washed with buffer A and placed in 30 ml of fresh supplemented Dulbecco’s modified Eagle’s medium. On day 4 the cells were harvested in 4 ml of ice-cold phosphate-buffered saline. They were collected by centrifugation at 2,000 x g for 10 min and washed once in phosphate-buffered saline. The final pellet was resuspended in 0.15 M KCl, 5 mM Tris-HCl, pH 7.2, and loosely homogenized. After centrifugation at 500 x g for 10 min to remove cell debris and nuclei, the supernatant was further centrifuged at 12,000 X g for 15 min to sediment mitochondria. A suspension of the latter in 0.15 M KCl, 15 mM Tris-HCl, pH 7.2, was assayed for CPT activity in the presence and absence of 50 pM malonyl-CoA, the difference between the two rates representing CPT I (1, 2, 10). A second aliquot was assayed after treatment with octyl glucoside (1% w/v) for 30 min at 0 “C to destroy CPT I activity and allow complete exposure of CPT II (1, 2, 10). A third sample was treated with Tween-20 (1% v/v), which also releases CPT II selectively from the membranes (1, 2). This extract was used for immunoprecipitation studies by the methods described in the preceding paper (1).

region within peptide 3 (5’-CCGTGNCCCATNGCN-

GCGTCGTT-3’). The probes were complementary to all

possible coding sequences for the amino acids represented. Approximately lo6 colonies of a rat liver cDNA library constructed in the vector pcDV were screened with the first oligonucleotide probe, yielding a single positive clone. Its identity was confirmed by hybridization with the second probe. This cDNA clone, -2.2 kilobases in length, was designated pCD-CPT II.1 (Fig. 1). Initial nucleotide analysis established that all three peptide sequences derived from the original protein could be predicted from the cDNA sequence. However, it seemed certain that the clone was not full length, for several reasons. First, the reading frame 5’ to the first potential ATG start codon contained no in frame termination codon. Second, based on analysis of Northern blots, the cDNA was 200-300 bases shorter than the CPT II mRNA length (see preceding paper). Third, and most important, the first potential start ATG coded for a methionine immediately preceding the first peptide sequenced (Met 43 in the complete sequence, Fig. 3). Miyazawa et al (8) had presented evidence that nascent CPT II has a mitochondrial targeting sequence that is cleaved in formation of the mature protein. Since Met 43 was presumably present in the purified mature enzyme, it could not have been the start of the targeting sequence.

To obtain the remaining sequence, a primer-extended cDNA library was constructed in the vector XgtlO. 106plaques were screened using a radiolabeled PstI to XhoI fragment (near the 5’ end of the first cDNA) as a probe. A single clone, XL4-CPT II.1 (Fig. l), was obtained. This 0.5kilobase cDNA contained an additional 78 bases 5’ to the 5’ terminus of the first cDNA clone. Nucleotide sequence analysis revealed that the reading frame of the original cDNA was now extended to

LLCCPT 11.1

Software-Alignment and analysis of the DNA and protein sequences were performed using IG-Suite and PC-Gene (Intelli- Genetics, Palo Alto, CA) and the University of Wisconsin Genetic Computer Group Programs (12).

Materials-Enzymes were from Boehringer Mannheim, New Eng- land Biolabs (Beverly, MA), Stratagene (LaJolla, CA), Promega

FIG. 1. Constructs used in the cloning and sequencing of rat liver CPT II cDNA. See text for details.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

cDNA for Carnitine Palmitoyltransferase II

include both an in frame initiation codon and an upstream, in frame termination codon.

The restriction map of the full-length cDNA, together with the sequencing strategies employed, are diagramed in Fig. 2. The complete cDNA sequence is shown in Fig. 3. It contains a 5’-untranslated stretch of 62 nucleotides, an open reading frame of 1,974 bases, predicting a 658 amino acid protein of 74,119 daltons, and the entire 3’-untranslated region of 260 nucleotides preceding the poly(A) tail. Although no strict consensus motif for a mitochondrial targeting peptide has yet been established, the first 30 amino acids have properties common to many mitochondrial leader sequences. There are

0 0.5 1.0 1.5 2.0 II"","","","",'?

W

--;- -

Q 2 2 r 2

L 1 2 2 ':

FE i g

1 pCD-CPT 11.1

=q -- -2.zi_-- e-r--=- - c m-- -e IL4-CPT 11.1 -

-7

FIG. 2. Restriction endonuclease map and sequencing strat- egy for rat liver CPT II cDNA. The stippled bar represents the coding reeion. Arrows under the solid bars indicate the extent and direction if the sequencing reactions.

FIG. 3. The complete sequence of rat liver CPT II cDNA. Nucleotides are numbered on the left, amino acids on the right. Squares and asterisks denote the stop codons in the 5’ non-coding region and at the 3’ end of the coding sequence, respectively. The arrow at nucleotide 17 shows the 5’ limit of the first clone, pCD-CPT 11.1. The amino acids underlined are those identified by automated Edman degradation of CNBr peptides derived from the original protein. They are referred to in the text by number beginning with the closest to the N terminus.

-62

1

91

181

271

351

451

541

631

721

811

901

$91

1981

21ocl

no acidic residues, four hydroxyl and four basic amino acids, and the remaining amino acids are mainly hydrophobic. A helical wheel plot (not shown) indicates that the serines and basic residues form one face of a putative helix, while the hydrophobic residues form the other face. (It is unlikely, however, that a continuous helical structure is formed, based on the frequency of proline residues within this stretch of amino acids).

Confirmation of the cDNA Identity-In order to confirm the identity of the cDNA clone, the first cDNA (pCD-CPT 11.1) was subcloned into the vector pBluescriptKS(+) to form pBKS-CPT II.3 (Fig. 1). In addition, a full-length cDNA was constructed in the same vector from the two original clones (pCD-CPT II.1 and XL4-CPT 11.1); this cDNA was designated pBKS-CPT II.4 (Fig. 1). The vector, pBluescript, contains both a T3 and a T7 promoter, which allows an RNA transcript of a cDNA to be synthesized in uitro. If a methylguanosine cap structure is incorporated into the RNA the result is a pseudo-mRNA that is capable of being translated. After in vitro transcription, both pBKS-CPT II.3 and pBKS-CPT II.4 yielded transcripts of the appropriate length (data not shown). When translated, each transcript produced an 35S-labeled protein that was specifically immunoprecipitated by anti-CPT II antibody, RK40. As seen from Fig. 4, the two products were of slightly different size. The shorter (lane 4), generated from

CCTGCTTGCTCAG66MCTCCCTCAGCCGCCGCGTTCTCTAGCC~CGTT~T~CCffi~CG . . .

llTG ATG CCG CGC CTG CTG TTT CGT 6CC T&i CCC CGC TGC CCC TCG CTT GTC CTG GGA GCC CCT AG, AGG CCC TTA b6T GCT 6TC TCG GGG kt Set Pro Arg Leu Leu Phe Arg Ala Trp Pro Arg Cyo Pro SW Le" "al Le" Gly Ala Pm Sar Arg Pr" Le" SW Ala "al Ser Gly

CCG GAC GA, TAT CTG C&G CAC AGC ATC GTG CCC Au: ATG CAC ;A; CA; FC AGC CT! ;;C AGG CTG CC, ATC CC, AM CT1 W 64'2 ACC Pro Psp Asp Tyr Le" Gln "ls SW Ile Val Pro Thr Wet li,s Y I" Set- Le o Ar" Le" Pro 11e PPO La Le" ClluAs" Thr 60

ATG FAG ,464 TAC CTC AA, GCA CAG LAG CCT CK TTG 6AT GRC AGC CA6 TTC AGG AGA #A 6M GCG TTG TGT I\AG MT TTT GAG ACT 6% Wet Lys Arg Tyr Le" Asn Ala Gln Lyr Pro Le" Le" Asp Asp Sar Gln Pk Arg Arg Thr 61" Ala Le" Cyr Lys As" Phe Gl" Thr Gly

GTT 666 PAG GAG CTG CAT GCC CAC CT6 CT, GCT CAG GAT MG CAG @AT MG CM ACC AGC TAC ATC TCA GGC CCC TGG TTC GA, ATG TAT "al Gly Lys Glu Lc" "Is Al., HI, Lc" Le" Ala Gln Asp Lys Gln Am Lys Hlr Thr Ser Tyr Ile Ser Gly Pro Trp Phe Asp Met Tyr

TTA ACT GCT CW. GAC TCT ATC GTT TTA MC TTT AA, CCA TTT AT6 6CA TTC MT CCG MC CCA AAG TCT GAG TAT MT W CAG CTT ACC Le" Thr Ala Arg Asp Ser Ile "a, Le" Asn Phe Am Pro Pk !4et Ala Phe Am Pro Asp Pro Lys Ser Gl" Tyr Asn Asp Gln Le" Thr

AGG GCA ACC MC TTG ACT GTT TC, GCA GTC CGG TTT CTG MG ACA CTT CAG GCT GGC CT, TTA GM CCA bAL GTG TTC CAC TTG MC CCT Arg Ala Thr As" Le" Thr "a, Ser Ala "a, kg Phe Le" Lyr Thr Le" Gln /\,a Gly Le" Le" G," Pro 6," "al Phe "4s Le" Am Pro

TCC MA AGT GAC AM GAC GCC TTC MA AGA CTC ATT CGC TTT GTT CCT CCC TCT CT6 TCC TGG TAT GGA GCC TIC CTG GTC MC GCA TAT Ser Lyr Ser Asp Thr Asp Ala Phe Ly9 Arg Le" Ile Arg Phe Val Pro Pro Ser Le" Ser Trp Tyr Gly Ala Tyr Le" Val Asn Ala Tyr

CCC CTG GRT ATG TCC CAG ,I\, TTC CGG CTT TTC AA, TCA ACT CGT ATA CCC A&& CCC AAT CGT GA, Gpsi CTC TTT ACT GAC ACC A&G GCT Pro Le" Asp "et Ser Gln Tyr Phe Arg Le" Phe At" Ser Thr Arg Ile Pro Arg Pro Asn Arg Asp Gl" Le" Phe Thr Asp Thr Lys Ala

AW CAC CTC CTG GTC CTA AW AAA GWI CAT TTC TAT GTC TTT UT GTC CTC GAT CM GA1 666 MC ATT GTG AAT CCC TTA GAG ATT CAG Arg "1, Le" Le" Val La" Arg Lyr Gly "II Phe Tyr Val Phe Asp Val La" Asp Gln Asp Gly As" 11s Val Air" Pro Le" Glu Ile Gln

GU CAT CT.4 AAG TAC ATT CTC TCA GAC AGC AGC CCT GTG CCC GAG TTT CC, GTG GCA TAT CTG KC AGT WG MC CGA GA1 GTC T66 GCA Ails Hlr Le" Lye Tyr Ile La" SW Isp Ser Se,' Pro Val Pro G," Phe Pro Va, Ala Tyr Leu Thr Ser Gl" Am Arg Asp Val Trp Ala

GAG CTC A&4 CAG MG TTG ATT TTT GAT GGC MT GAG MA KC CTG AAG &AA GTG 6AC TCT GCT 6TC TTC TGC CTC TGC CTA GA, MC TTC G,,, Le" Arg Gin Lys Le" Ile Phe Asp Gly As" Gl" Gl" Thr Le" Lys Lys Val Asp SW Ala Val Phe Cys Le" Cys Le" Asp Asp Phs

",41 f;:; MG y CTT A;, fA; ;I" TCA CAC ACC ATG CTG CAC G&C GAT 66C AC.4 MC CGC T66 TTT GAT MG TCC TTT MC CTC ATT GTA LYJ 0 Le" 1 e u SF, HIS Thr Met Le" H,s G,y Asp Gly Thr Aisn Arg Trp Phe Asp Lys Ssr Phe As" La" Ile Val

EC GAG GAC GGC ACT 6CT GCA WC CRC TTC GAG CM TCG TGG GGG GA, 666 GTG 6% GTG CT, AGG TTT TIC MT 6t.A GTG TTC MA GAC Ala Glu Asp Gly Thr Ala Ala Va, ",s Phe Gl" "5s Ser Trp Gly Asp Gly Val Ala Val Le" Arg Pk Phe Am Gl" Val Pk Arg Asp

AGC ACT GIG KC CC, GCC ,,TC ACT CCC CAG AGC CAG CC, GCC GCC ACC MC TCC TCT GCG TC, GTG GAG KG UC AW TTC UC CTC AGC Ser Thr Gin Thr Pro Ala ,,e Thr Pro G,n SW Gln Pro Ala Ala Thr As" Ser Ser Ala SW Val Glu Thr Le" SW Phe Am Le" SW

GGT GCT CTC MG GCT GGC ATC ACT GCT GCC AAG GAG MG TTC 6.K KC ACG GTG AM ACC CTC MC ATT GAC TCC ATT CA6 TTC CAG A64 Gly Ala Le" Lys Ala Gly Ile Thr Ala ,.,a Lys Gl" Lyr Phe Asp Thr Thr Val Lyo Thr LB" SW Ile Asp Ser Ile Gln Pk Gln Arg

IT GGC FAG GAG TTC CTG AAG AAG FAG WIG CTG AGC CCC WT GCG GTG GCC CAG CTG 6CC TTC CA.6 ATG GCC TTC CTG A6A CA6 TAT GGC GIy Gly Lys G," Phe Le" Lyr Lys Lys Gin Le" Ssr Pro Asp Ala Va, Ala Gln Le" Ala Phe Gln "et Ala Phe Lc" Arg Gln Tyr Gly

CAG ACG GTG GCT ACC TAT GkG KC T'X AGC ACT GCA GCC TIC RAG CAC 6% CGC AC.4 GAG ACT AK CGC CCA GCC ICC ATC ill ACT AAG Gin Thr Val kls Thr Tyr Gl" Se, Cyr Ser Thr Ala Ala Phe Lys "lr Gly Arg Thr Glu Thr Ile Arg Pro Ala Ser tie Pk Thr LYS

AGA TM: TCC GAG GCG TTT GTC 1\GG MC CCC TCC AAG CAC AGT 6TG GGZ WG CT, CAG CAC ATG ATG GCT "f" TG, TCC MA TAC CAT G6C I\rg Cyo Ser Glu Ala Phe ',a, Arg Asp Pro SW Lys "1s Ser Val Gly Gl" Le" 61" His Met Meet 1 Aa

CAG CT:, 1;; AA.4 $A GCA G;z ii," G$ CA," G$ ;;J GAC CGA CAC TTG ;"; FT CT: ;4" TIT CT: ;?A ;,"; GfC AGA Gr CTC MC CTA 5," Le LVS " Ala A G Y G Y ASD Arc, His Le" Y ,a Le 9 Tvr Le a A o Ars G Y Leu Am Lw

;;T f33 CTC TAT CTG GAT CC, GCA TAC CAG CAG ATG MC CAC MC ATC CT6 TCC KC AGC ACT CTG MC AM: CCA 6CA GTG AK CTT GGG o " Le" TVr Le" Asp Pro Ala Tyr Gln 6," Het Am Hlr Ax Ile Le" Ser Thr Ser Thr Le" Asn Ser Pro Ala Val SW Le" Gly

GGC TTT GCC CCT GTG GTC CC, GAT GGC TTT GGC ATT GCA TAT GC, GTT CAC MT GAC TGG AT1 64C TGC MT GTC TCC TCC TIC TCA 664 G,y Phe Ala Pro Val Va, Pro A,p Gly Phe Gly Ile Ala Tyr Ala Val "11 Asp Asp Trp Ile Gly Cys Am Va, SW Ser Tyr Ser 61~

CGC A&T GCC CGA 6AG TTT CTC CAC TGT WC CAG AAG T6C TTG GM 6AC ATT TTC GAT GCT CTA GM GGC APA GCC ATC MA ACT TAG CT1 Arg Am Ala Arg Gl" Phe Le" H,o Cys Val Gln Lys Cys Le" Gl" Asp Sk Phe Asp Ala Le" Gl" GIy Lyr Ala IIe Lyr Thr l **

30

90

120

150

180

210

240

270

300

330

360

390

420

450

480

510

540

570

600

630

653

2219 ACCTGGTGTT6CTTAG~Aln


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


RNA - - P3 P3 P4 P4 RK39 + - + - + - RK40 - + - + - +

12 3 4 56 116-

97- m b 66- ;; 5

43-

- ~‘. +CPT II

FIG. 4. Immunoblot of transcription/translation products. The short and full-length cDNAs subcloned into the vector pBluescriptKS(+), pBKS-CPT II.3 and 4 (abbreviated to p3 and p4, respectively), were used for in vitro transcription and translation. The reaction mixtures were then treated with preimmune (RK39) or immune (RK40) IgG raised against purified CPT II. Immune complexes precipitated with protein A were analyzed by SDS-PAGE followed by fluorography. Lanes 1 and 2 contained no added RNA in the translation step. The migration position of pure CPT II is shown on the right.

TABLE I CP7’ activity in mitochondria from transfected COS cells

COS cells were transfected with the indicated species of DNA as described under “Experimental Procedures.” After 4 days the cells were disrupted and the mitochondria-rich fraction was assayed for CPT I and CPT II activitv.

DNA used for transfection

CPT activity

CPT I CPT II

None Salmon sperm pCMV4 vector pCMV4-CPT II. 4

nmol. mm-‘. mgproteu-’ 0.24 1.38 0.24 1.41 0.33 1.35 0.20 7.72

pBKS-CPT 11.3, migrated to a position fractionally below that of pure CPT II, suggesting that translation began at the first available AUG codon, presumably Met 43 (Fig. 3). By contrast, the product from pBKS-CPT II.4 (lane 6) was slightly larger than the pure enzyme, consistent with translation having begun at Met 1.’ It seemed likely that this constituted the same protein as that derived from pBKS-CPT II.3 but containing, in addition, a mitochondrial targeting sequence. (Note, however, that both products must have lacked the carboxyl-terminal7 amino acid residues of mature CPT II since the constructs used for transcription had been linearized with restriction endonuclease, X&I (Fig. 2).)

As a second approach to establishing the clone’s identity, a peptide corresponding to the predicted residues 223 to 242 in Fig. 3 was synthesized and, after conjugation to purified protein derivative of tuberculin, was used to raise an antibody in a rabbit. In experiments not shown the immune serum clearly reacted with authentic CPT II on Western blot analysis. Preimmune serum from the same animal gave no signal.

The most direct evidence that the cDNA of pBKS-CPT II.4 encodes CPT II came from DNA transfection experiments in which the full-length cDNA was introduced into COS cells. A typical result is shown in Table I which illustrates three points. First, mitochondria from these cells contain both CPT I and II activity (the former was completely inhibitable by malonyl-CoA, the latter insensitive). Second, the level of CPT I was unaffected by any of the DNA species used to transfect the cells. Third, CPT II activity was uniquely induced, by a factor of almost B-fold, in cells transfected with pCMV4-CPT

’ Note that the ATG triplets corresponding to Met 1 and Met 43 in Fig. 3 are both preceded by nucleotide sequences that conform to the consensus motif for an initiator condon as defined by Kozak (13).

TABLE II

Immunoprecipitation of COS cell CPT II activity by antibody RK40 The mitochondria-rich fraction from each group of COS cells used

in the experiment of Table I was treated with Tween-20 (1% v/v). The extract, containing all of the original CPT II activity, was then subjected to immunoprecipitation using preimmune antibody RK39 or anti-rat liver CPT II antibody RK40, followed by Pansorbin as described previously (1). Enzyme activity remaining in the final supernatant is expressed as a percent of that found when no antibody was used.

DNA used for 96 of mitochondrial CPT II

activity remaining after transfection

No antibody RK39 RK40

None 100 87 0 Salmon sperm 100 98 0 pCMV4 vector 100 87 0 nCMV4-CPT II. 4 100 91 0

__ 40 Hydrophilic

-50 ~~,,~,~~,‘,,~~,,~,,‘,~~,-,““~~““,,I’1’(””,I~~,~~‘,~“““( 0 100 200 300 400 500 600

Amino kid Number

FIG. 5. Hydropathy plot for rat liver CPT II. The profile was generated using the method of Kyte and Doolittle (14) with an interval of nine amino acids.

CFTII COT CMT

CPTII COT CMT

. . . . . . . . . . . . . . . . . . . . . EALWV@gTo~PD@LLLLKDAIRA9

FIG. 6. Alignment of predicted amino acid sequences for three acyltransferases. The deduced amino acid sequence for rat liver mitochondrial CPT II has been aligned with those for rat liver peroxisomal carnitine octanoyltransferase (COT) and porcine choline acetyltransferase (ChA77 using computer program GAP (12). Shaded boxes indicate sequence identity between CPT and one or both of the other proteins. Sequence identity between rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase is not highlighted.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

10724 cDNA for Carnitine Palmitoyltransferase II

11.4. Moreover, both the induced enzyme and the endogenous CPT II were completely immunoprecipitated by antibody RK40 (Table II).

The Kyte-Doolittle hydrophobicity plot for the predicted amino acid sequence of CPT II is depicted in Fig. 5. Curiously, from computer analysis employing a number of programs in the software package, PC-Gene, no particular region of the molecule was predicted to be membrane spanning. However, it is recognized that in such programs the criteria used for a positive prediction are somewhat rigid and frequently miss known transmembrane domains (15).

The predicted sequences of two other acyl-CoA utilizing enzymes, rat liver peroxisomal carnitine octanoyltransferase) and porcine choline acetyltransferase have recently been pub- lished (16, 17). Computer alignment of the deduced amino acid sequence of CPT II with those of rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase (Fig. 6) revealed that CPT II possessed 27 and 25% identity with rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase, respectively. When allowance is made for conservative changes of amino acids these values increase to 51 and 53% similarity, respectively.

DISCUSSION

To begin to explore the mitochondrial CPT system at a structural level, we set out to determine the primary amino acid sequence of one of its component enzymes, CPT II. This protein was chosen because we already had sufficient purified material from rat liver (2) with which to obtain partial amino acid sequences to initiate cloning procedures. Using classical strategies we succeeded in isolating and sequencing what appeared to be a cDNA corresponding to the entire coding region of the protein. Confidence in its identity comes from the following considerations. First, the nucleotide sequence, in a single open reading frame, predicted all three of the peptides found in the original protein. Second, an antibody raised against a fourth peptide composed of a stretch of 20 amino acids predicted by the cDNA sequence recognized pure CPT II on a Western blot. Third, the cDNA, pBKS-CPT 11.4, as well as a shorter construct lacking 78 base pairs at the 5’ end (pBKS-CPT 11.3), were transcribed and translated in uitro, yielding products that were selectively precipitated by anti-CPT II antibody. Based on SDS-PAGE analysis, these proteins were slightly larger and smaller, respectively, than mature CPT II. This would be consistent with the notion that in the case of pBKS-CPT II.4 translation of the message began at the AUG codon representing Met-l in Fig. 3, whereas Met-43 was the probable start site on the shorter transcript. Fourth, and most importantly, transfection of COS cells with the full-length cDNA ligated into the vector, pCMV4, resulted in a 6-fold increase in mitochondrial CPT II activity that was also immunoprecipitated by the anti-CPT II antibody.3

’ In a series of reports culminating with Ref. 18, the laboratory of Brady and Brady have used an oligonucleotide with the stated se- ouence: CT(C.T)TT(C.T)CC(G,A,T,C)CA(G,A,T,C)GT(C,T)TG, to detect mRNA for rat liver CPT. If, as seems likely, the authors were initiallv dealing with mitochondrial CPT II (i.e. the readily purified, catalytically active enzyme with M, - 70 kDa), several problems arise. First, the oligonucleotide was based on the amino acid sequence: pyro- Glu-Lys-Gly-Val-Gln-Thr-Gly-Gln, which they believe to constitute the N terminus of the protein (19). This sequence is not represented in our clone (Fig. 3). Second, we must assume that the oligonucleotide depicted above was inadvertently written in the 3’ to 5’, rather than the conventional 5’ to 3’ direction, since only the former would be appropriate for hybridization to mRNA encoding the peptide sequence given (19). Because of the ambiguity, we synthesized the 17- base oligonucleotide corresponding to both possibilities, i.e. taking the C (left terminus) or the G (right terminus) as the 5’ base. After

Inspection of the predicted amino acid sequence of CPT II (Fig. 3) reveals that the N-terminal segment preceding Asp- 32 has the general characteristics of a mitochondrial leader peptide (20). Based on the data of Miyazawa et al (8) and Fig. 4, it seems likely that a significant portion of the targeting sequence is cleaved upon mitochondrial import of the protein, thus reducing its size from the predicted value of 74,119 daltons to that of mature CPT II which is generally taken as -70 kDa (21; but see footnote 4 in Ref. 1).

Available evidence indicates that unlike CPT I, which appears to be deeply anchored in the mitochondrial outer membrane, CPT II is less tightly associated with its (inner) membrane environment (2, 21). Although no strong predictions for transmembrane regions emerged from computer analysis, visual inspection of the hydrophobicity plot for CPT II (Fig. 5) and of the deduced amino acid sequence (Fig. 3) suggests the possible presence of a limited number of membrane spanning domains. One might be within the hydrophobic peak in the vicinity of amino acid 600. Studies are in progress to define the precise topographical relationship between membrane and enzyme.

Not surprisingly, when the nucleotide sequence for rat liver mitochondrial CPT II was compared with the GenBank and EMBL computer data bank, the two proteins with which it proved to be most similar were both of the acyltransferase class. These were rat liver carnitine octanoyltransferase (COT) and porcine choline acetyltransferase (ChAT), both of whose sequences have recently been reported (16, 17). CPT II was found to be identical over some 25-30% of its residues with each of these proteins and, allowing for conservative amino acid changes, the degree of similarity between CPT II and each of the other transferases appears to be -50%. It seems reasonable to suppose that the binding sites for the substrates of CPT II, namely, acyl-CoA, CoA, carnitine, and acylcarnitine, will be found to reside within these regions of similarity between CPT II and COT and/or ChAT.

In summary, the data presented provide the first insight into the primary structure of a mitochondrial isozyme of carnitine palmitoyltransferase. Although further studies are needed to determine the mechanism by which the nascent protein gains entry into the mitochondrion, where its targeting sequence is cleaved, and how the mature enzyme associates with the inner membrane, the present findings provide a first step toward these goals. They should also prove valuable in the analysis of CPT II isoforms and their genes in different tissues and species. Such knowledge will be indispensable in the quest to understand the genetic basis of the human CPT II deficiency syndromes.

Acknowledgments-We are indebted to Murphy Daniels and Petra Contreras for their invaluable technical assistance, and to Carolyn R. Moomaw and Lynn De Ogny for performing automated amino acid sequence analyses.

REFERENCES

1. Woeltje, K. F., Esser, V., Weis, B. C., Cox, W. F., Schroeder, J. G.. Liao, S-T.. Foster. D. W., and McGarry, J. D. (1990) J. Bid. Ch&n. 2$5, 10714-10719

2. Woeltje, K. F., Kuwajima, M., Foster, D. W., and McGarry, J. D. (1987) J. Biol. Chem. 262, 9822-9827

3. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular

end labeling with “P, both were tested for hybridization to construct pBKS-CPT II.4 on dot blots. Neither gave a positive signal, whereas a 20-mer oligonucleotide complementary to bases 127-146 in our clone reacted intensely. While we have no explanation for the dis- crepancy, for reasons given in the text we feel confident about the validity of the cDNA sequence depicted in Fig. 3.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

4.

5.

6.

7. 8.

9.

10.

11.


Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Sanger, F., Nicklen, S., and Coulson, A. R. (1977) hoc. Natl. Acad. Sci. U. S. A. 74,5463-5467

Tabor, S., and Richardson, C. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,4767-4771

MacDonald, R. J., Swift, G. H., Przybyla, A. E., and Chirgwin, J. J. (1987) Methods Enzymol. 152,219~226

McPhaul, M., and Berg, P. (1987) Mol. Cell. Biol. 7, 1841-1847 Miyazawa, S., Ozasa, H., Osumi, T., and Hashimoto, T. (1983) J.

Biochem. (Tokyo) 94, 529-542 Andersson, S., Davis, D. L., Dahlback, H., Jornvall, H., and

Russell, D. W. (1989) J. Biol. Chem. 264, 8222-8229 Declercq, R. E., Falck, J. R., Kuwajima, M., Tyminski, H., Foster,

D. W., and McGarry, J. D. (1987) J. Biol. Chem. 262, 9812- 9821

Gorman, C. (1985) in DNA Cloning ZZ: A Practical Approach (Glover, D. M., ed) pp. 143-190, IRL Press, Washington, D. C.

12.

13. 14. 15.

16.

17.

18.

19.

20.

21.

Devereux, J., Haeberli, P., and Smithies, 0. (1984) Nucleic Acids Res. 12, 387-395

Kozak, M. (1984) Nucleic Acids Res. 12, 857-872 Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 Nishikawa, K., and Ooi, T. (1986) Biochim. Biophys. Acta 871,

45-54 Chatterjee, B., Song, C. S., Kim, J. M., and Roy, A. K. (1988)

Biochemktry27,9000-9006 Berrard, S., Brice, A., Lottspeich, F., Braun, A., Barde, Y-A., and

Mallet, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,9280-9284 Wang, L., Brady, P. S., and Brady, L. J. (1989) Biochem. J. 263,

703-708 Brady, P. S., Feng, Y-X, and Brady, L. J. (1988) J. Nutr. 118,

1128-1136 Lemire, B. D., Fankhauser, C., Baker, A., and Schatz, G. (1989)

J. Biol. Chem. 264, 20206-20215 McGarry, J. D., Woeltje, K. F., Kuwajima, M., and Foster, D. W.

(1989) Diabetes Metab. Rev. 5, 271-284


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Foster and J D McGarryK F Woeltje, V Esser, B C Weis, A Sen, W F Cox, M J McPhaul, C A Slaughter, D W

carnitine palmitoyltransferase II.Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial

1990, 265:10720-10725.J. Biol. Chem.

http://www.jbc.org/content/265/18/10720Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/265/18/10720.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/265/18/10720

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;265/18/10720&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/265/18/10720

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=265/18/10720&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/265/18/10720

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/265/18/10720.full.html#ref-list-1

http://www.jbc.org/

Cloning, Sequencing, and Expression of a cDNA Encoding Rat ...Printed in U.S.A. Cloning, Sequencing,...

Documents

Transcript of Cloning, Sequencing, and Expression of a cDNA Encoding Rat ...Printed in U.S.A. Cloning, Sequencing,...