ABCMdb

PMID: 17761678

Kashiwayama Y, Asahina K, Morita M, Imanaka T
Hydrophobic regions adjacent to transmembrane domains 1 and 5 are important for the targeting of the 70-kDa peroxisomal membrane protein.
J Biol Chem. 2007 Nov 16;282(46):33831-44. Epub 2007 Aug 30., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln In the NH2-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1.Bythedisruptionofthesehydrophobicmotifsbythemuta- tionofL21Q/L22Q/L23QorI70N/L71Q,PMP70(AA.1-144)-GFP lost targeting efficiency. Login to comment 88 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Lys120Ala ABCD3 p.Leu23Ala ABCD3 p.Arg124Ala ABCD3 p.Lys53Ala ABCD3 p.Arg117Ala ABCD3 p.Lys61Ala ABCD3 p.Leu23Gln ABCD3 p.Arg77Ala ABCD3 p.Ile70Asn ABCD3 p.Pro76Ala ABCD3 p.Ile70Ala ABCD3 p.Leu308Gln ABCD3 p.Lys28Ala ABCD3 p.Arg119Ala ABCD3 p.Leu21Ala ABCD3 p.Lys38Ala ABCD3 p.Lys123Ala ABCD3 p.Lys54Ala ABCD3 p.Arg29Ala ABCD3 p.Leu71Ala ABCD3 p.Arg31Ala ABCD3 p.Leu22Ala ABCD3 p.Arg66Ala ABCD3 p.Lys72Ala ABCD3 p.Ile307Asn ABCD3 p.Arg30Ala ABCD3 p.Arg56Ala ABCD3 p.Lys39Ala The lysate was cen- TABLE 2 Oligonucleotide primer sequences used for the generation of mutant PMP70 constructs Conctruct name Forward primer (5؅ 3 3؅) K28A/R29A GCTCTGCCTGCTCCACGCGGCGCGCCGCGCCCTCG R30A/R31A CCTGCTCCACGCGGCGGCCGCCGCCCTCGGCCTGCACG K38A/K39A CCTCGGCCTGCACGGTGCGGCAAGTGGAAAACCACCATTAC P76A/R77A CAGATTCTGAAAATCATGGTCGCTGCAACATTTTGTAAAGAGACAGG K72A GGCTCATACAGATTCTGGCAATCATGGTCGCTGCAAC R66A GGACAAGGTGTTTTTCTCAGCGCTCATACAGATTCTGGC K61A CGAGCTGTGGTGGACGCGGTGTTTTTCTCAGC K53A/K54A/R56A CAATGAGAAAGAGGGGGCAGCAGAGGCAGCTGTGGTGGACGCGG K123A/R124A GGTCGTAGCAGGAAAGATTTCGCGGCATACTTACTCAACTTCATCG R119A/K120A GGTATCATTGGTCGTAGCGCGGCAGATTTCGCGGCATACTTACTC R117A GTGGTATCATTGGTGCTAGCGCGGCAGATTTCGCG L21A/L22A/L23A GTGCCGCGTTCGCTGCTGCTTGCCTGCTCCAC L21Q/L22Q/L23Q GCTGGTGCCGCGTTCCAGCAGCAGTGCCTGCTCCACAAGC I70A/L71A CTCAAGGCTCATACAGGCTGCGAAAATCATGGTCCCTAGAAC I70N/L71Q CTCAAGGCTCATACAGAATCAGAAAATCATGGTCCCTAGAAC I307N/L308Q GGTGGAACACCTACATAATTTCAATCAGTTTCGGTTTTCAATGGGC Peroxisome Targeting Signal of PMP70 NOVEMBER 16, 2007•VOLUME 282•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 33833 trifuged at 20,000 ϫ g for 30 min and the His-Pex19p in the supernatant was immediately applied to 10 ml of TALON Metal affinity resin (Clontech) equilibrated with the lysis buffer. Login to comment 89 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Lys120Ala ABCD3 p.Leu23Ala ABCD3 p.Arg124Ala ABCD3 p.Lys53Ala ABCD3 p.Arg117Ala ABCD3 p.Lys61Ala ABCD3 p.Leu23Gln ABCD3 p.Arg77Ala ABCD3 p.Ile70Asn ABCD3 p.Pro76Ala ABCD3 p.Ile70Ala ABCD3 p.Leu308Gln ABCD3 p.Lys28Ala ABCD3 p.Arg119Ala ABCD3 p.Leu21Ala ABCD3 p.Lys38Ala ABCD3 p.Lys123Ala ABCD3 p.Lys54Ala ABCD3 p.Arg29Ala ABCD3 p.Leu71Ala ABCD3 p.Arg31Ala ABCD3 p.Leu22Ala ABCD3 p.Arg66Ala ABCD3 p.Lys72Ala ABCD3 p.Ile307Asn ABCD3 p.Arg30Ala ABCD3 p.Arg56Ala ABCD3 p.Lys39Ala The lysate was cen- TABLE 2 Oligonucleotide primer sequences used for the generation of mutant PMP70 constructs Conctruct name Forward primer (5d15; 3 3d15;) K28A/R29A GCTCTGCCTGCTCCACGCGGCGCGCCGCGCCCTCG R30A/R31A CCTGCTCCACGCGGCGGCCGCCGCCCTCGGCCTGCACG K38A/K39A CCTCGGCCTGCACGGTGCGGCAAGTGGAAAACCACCATTAC P76A/R77A CAGATTCTGAAAATCATGGTCGCTGCAACATTTTGTAAAGAGACAGG K72A GGCTCATACAGATTCTGGCAATCATGGTCGCTGCAAC R66A GGACAAGGTGTTTTTCTCAGCGCTCATACAGATTCTGGC K61A CGAGCTGTGGTGGACGCGGTGTTTTTCTCAGC K53A/K54A/R56A CAATGAGAAAGAGGGGGCAGCAGAGGCAGCTGTGGTGGACGCGG K123A/R124A GGTCGTAGCAGGAAAGATTTCGCGGCATACTTACTCAACTTCATCG R119A/K120A GGTATCATTGGTCGTAGCGCGGCAGATTTCGCGGCATACTTACTC R117A GTGGTATCATTGGTGCTAGCGCGGCAGATTTCGCG L21A/L22A/L23A GTGCCGCGTTCGCTGCTGCTTGCCTGCTCCAC L21Q/L22Q/L23Q GCTGGTGCCGCGTTCCAGCAGCAGTGCCTGCTCCACAAGC I70A/L71A CTCAAGGCTCATACAGGCTGCGAAAATCATGGTCCCTAGAAC I70N/L71Q CTCAAGGCTCATACAGAATCAGAAAATCATGGTCCCTAGAAC I307N/L308Q GGTGGAACACCTACATAATTTCAATCAGTTTCGGTTTTCAATGGGC Peroxisome Targeting Signal of PMP70 NOVEMBER 16, 2007ߦVOLUME 282ߦNUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 33833 trifuged at 20,000 afb; g for 30 min and the His-Pex19p in the supernatant was immediately applied to 10 ml of TALON Metal affinity resin (Clontech) equilibrated with the lysis buffer. Login to comment 128 ABCD3 p.Lys120Ala ABCD3 p.Arg124Ala ABCD3 p.Arg117Ala ABCD3 p.Lys61Ala ABCD3 p.Arg77Ala ABCD3 p.Lys28Ala ABCD3 p.Arg119Ala ABCD3 p.Lys38Ala ABCD3 p.Lys123Ala ABCD3 p.Lys54Ala ABCD3 p.Arg29Ala ABCD3 p.Arg31Ala ABCD3 p.Arg66Ala ABCD3 p.Lys72Ala ABCD3 p.Arg30Ala ABCD3 p.Arg56Ala ABCD3 p.Lys39Ala As shown in Fig. 3B, PMP70(AA.1-144 K28A/R29A/R30A/ R31A/K38A/K39A)-GFP,PMP70(AA.1-144K53A/K54A/R56A/ K61A/R66A/K72A/R77A)-GFP, and PMP70(AA.1-144 R117A/ R119A/K120A/K123A/R124A)-GFP were localized to peroxisomes, and the targeting efficiency of each mutant protein was almost the same as that of PMP70(AA.1-144)-GFP. Login to comment 129 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Lys120Ala ABCD3 p.Arg124Ala ABCD3 p.Arg117Ala ABCD3 p.Lys61Ala ABCD3 p.Leu23Gln ABCD3 p.Arg77Ala ABCD3 p.Ile70Asn ABCD3 p.Lys28Ala ABCD3 p.Arg119Ala ABCD3 p.Lys38Ala ABCD3 p.Lys123Ala ABCD3 p.Lys54Ala ABCD3 p.Arg29Ala ABCD3 p.Arg31Ala ABCD3 p.Arg66Ala ABCD3 p.Lys72Ala ABCD3 p.Arg30Ala ABCD3 p.Arg56Ala ABCD3 p.Lys39Ala Under the same condition, PMP70(AA.1-144 L21Q/L22Q/L23Q)- GFP and PMP70(AA.1-144 I70N/L71Q)-GFP were not localized to peroxisomes as negative controls (see Fig. 4). Login to comment 130 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn Under the same condition, PMP70(AA.1-144 L21Q/L22Q/L23Q)- GFP and PMP70(AA.1-144 I70N/L71Q)-GFP were not localized to peroxisomes as negative controls (see Fig. 4). Login to comment 132 ABCD3 p.Thr83Ala ABCD3 p.Leu86Ala ABCD3 p.Glu82Ala ABCD3 p.Gly113Val ABCD3 p.Leu88Ala ABCD3 p.Gly84Ala ABCD3 p.Ile68Ala ABCD3 p.Phe63Ala ABCD3 p.Leu67Ala ABCD3 p.Arg96Ala ABCD3 p.Tyr85Ala ABCD3 p.Phe20Val ABCD3 p.Ser95Ala ABCD3 p.Ala19Val ABCD3 p.Ala18Val ABCD3 p.Ile114Ala ABCD3 p.Val62Ala We replaced these amino acids with Ala or Val in PMP70(AA.1-144)-GFP as follows: A18V/A19V/F20V, V62A/F63A, L67A/I68A, E82A/ T83A, G84A/Y85A/L86A/L88A, S95A/R96A, and G113V/ I114A, respectively. Login to comment 133 ABCD3 p.Thr83Ala ABCD3 p.Leu86Ala ABCD3 p.Glu82Ala ABCD3 p.Gly113Val ABCD3 p.Leu88Ala ABCD3 p.Gly84Ala ABCD3 p.Ile68Ala ABCD3 p.Phe63Ala ABCD3 p.Leu67Ala ABCD3 p.Arg96Ala ABCD3 p.Tyr85Ala ABCD3 p.Phe20Val ABCD3 p.Ser95Ala ABCD3 p.Ala19Val ABCD3 p.Ala18Val ABCD3 p.Ile114Ala ABCD3 p.Val62Ala We replaced these amino acids with Ala or Val in PMP70(AA.1-144)-GFP as follows: A18V/A19V/F20V, V62A/F63A, L67A/I68A, E82A/ T83A, G84A/Y85A/L86A/L88A, S95A/R96A, and G113V/ I114A, respectively. Login to comment 135 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn To examine whether these hydrophobic motifs are important for the targeting of PMP70, we disrupted these hydrophobic properties by the mutation of L21Q/L22Q/L23Q or I70N/L71Q and examined the subcellular localization. Login to comment 136 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Ala ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Leu21Ala ABCD3 p.Leu71Ala ABCD3 p.Leu22Ala When these amino acid residues were substituted for alanines considered to retain the minimum hydrophobic property of these sequences in PMP70(AA.1-144)-GFP, PMP70(AA.1-144 L21A/L22A/L23A)- GFPandPMP70(AA.1-144I70A/L71A)-GFPwerebothlocalized to peroxisomes (Fig. 4A). Login to comment 137 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Ala ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Leu21Ala ABCD3 p.Leu71Ala ABCD3 p.Leu22Ala On the other hand, we could not detect the fluorescence of PMP70(AA.1-144 L21Q/L22Q/L23Q)- GFP or PMP70(AA.1-144 I70N/L71Q)-GFP. Login to comment 138 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn In addition, PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP and PMP70(AA.1-144 I70N/L71Q)-GFP were not detected by immunoblot analysis under the conditions in which PMP70(AA.1-144)-GFP was detected (Fig. 4B, left). Login to comment 139 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn However, in the presence of MG132, a proteasome inhibitor, degradations of these mutant proteins were inhibited, and both PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP and PMP70(AA.1-144 I70N/L71Q)-GFP exhibited almost the sameexpressionlevelsasthatofPMP70(AA.1-144)-GFP(Fig.4B, right). Login to comment 140 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn In this condition, PMP70(AA.1-144)-GFP was still localized to peroxisomes, but PMP70(AA.1-144 L21Q/L22Q/L23Q)- GFP and PMP70(AA.1-144 I70N/L71Q)-GFP did not show any peroxisomal localization (Fig. 4C). Login to comment 141 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn Furthermore, full-length PMP70 with the same mutations (PMP70(AA.1-659 L21Q/ Peroxisome Targeting Signal of PMP70 NOVEMBER 16, 2007•VOLUME 282•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 33835 PMP70(AA.1-124/TMD4)-GFP PMP70(AA.1-124/TMD6)-GFP PMP70(TMD2)-GFP PMP70(TMD4)-GFP PMP70(TMD6)-GFP PMP70(AA.1-124)-GFP PMP70(AA.1-144)-GFP (B) Peroxisomes GFP Superimposition Peroxisomal localization + - - - - + + 2 113 144 PMP70(TMD2)-GFP 259 4 224 PMP70(TMD4)-GFP 6 314 347 PMP70(TMD6)-GFP 1 259 4 224124 1 1 PMP70(AA.1-124/TMD4)-GFP 124 1 1 6 314 347 PMP70(AA.1-124/TMD6)-GFP 124 1 1 PMP70(AA.1-124)-GFP 1441 1 2PMP70(AA.1-144)-GFP (A) FIGURE 2. Login to comment 142 ABCD3 p.Leu21Gln Furthermore, full-length PMP70 with the same mutations (PMP70(AA.1-659 L21Q/ Peroxisome Targeting Signal of PMP70 NOVEMBER 16, 2007ߦVOLUME 282ߦNUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 33835 PMP70(AA.1-124/TMD4)-GFP PMP70(AA.1-124/TMD6)-GFP PMP70(TMD2)-GFP PMP70(TMD4)-GFP PMP70(TMD6)-GFP PMP70(AA.1-124)-GFP PMP70(AA.1-144)-GFP (B) Peroxisomes GFP Superimposition Peroxisomal localization + - - - - + + 2 113 144 PMP70(TMD2)-GFP 259 4 224 PMP70(TMD4)-GFP 6 314 347 PMP70(TMD6)-GFP 1 259 4 224 124 1 1 PMP70(AA.1-124/TMD4)-GFP 124 1 1 6 314 347 PMP70(AA.1-124/TMD6)-GFP 124 1 1 PMP70(AA.1-124)-GFP 144 1 1 2 PMP70(AA.1-144)-GFP (A) FIGURE 2. Login to comment 145 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu23Gln Peroxisome Targeting Signal of PMP70 33836 L22Q/L23Q)-GFPandPMP70(AA.1-659I70N/L71Q)-GFP)was not localized to peroxisomes either. Login to comment 146 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu23Gln Peroxisome Targeting Signal of PMP70 33836 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282ߦNUMBER 46ߦNOVEMBER 16, 2007 at SEMMELWEIS UNIV OF MEDICINE on December 4, L22Q/L23Q)-GFPandPMP70(AA.1-659I70N/L71Q)-GFP)was not localized to peroxisomes either. Login to comment 152 ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln On the other hand, PMP70(AA.1-659 L21Q/L22Q/L23Q) was not well retained in a soluble form, and only 25% of PMP70(AA.1-659 L21Q/ L22Q/L23Q) was recovered in the soluble fraction even in the presence of His-Pex19p. Login to comment 153 ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln On the other hand, PMP70(AA.1-659 L21Q/L22Q/L23Q) was not well retained in a soluble form, and only 25% of PMP70(AA.1-659 L21Q/ L22Q/L23Q) was recovered in the soluble fraction even in the presence of His-Pex19p. Login to comment 154 ABCD3 p.Leu71Gln ABCD3 p.Lys120Ala ABCD3 p.Arg124Ala ABCD3 p.Lys53Ala ABCD3 p.Arg117Ala ABCD3 p.Lys61Ala ABCD3 p.Arg77Ala ABCD3 p.Ile70Asn ABCD3 p.Pro76Ala ABCD3 p.Lys28Ala ABCD3 p.Arg119Ala ABCD3 p.Lys38Ala ABCD3 p.Lys123Ala ABCD3 p.Lys54Ala ABCD3 p.Arg29Ala ABCD3 p.Arg31Ala ABCD3 p.Arg66Ala ABCD3 p.Lys72Ala ABCD3 p.Arg30Ala ABCD3 p.Arg56Ala ABCD3 p.Lys39Ala PMP70(AA.1-659 I70N/L71Q) existed in a soluble form in the presence of His-Pex19p and still interacted with His-Pex19p at almost the same level as wild type PMP70 did, but the mutant PMP70 was mislocalized to endoplasmic reticulum-like structures, as (B) (A) PMP70(AA.1-144 K28A/R29A/R30A/R31A/K38A/K39A)-GFP PMP70(AA.1-144 K53A/K54A/R56A/K61A/R66A/K72A/P76A/R77A)-GFP PMP70(AA.1-144 R117A/R119A/K120A/K123A/R124A)-GFP MAAFSKYLT-----------ARNSS-LAGAAFLL--LCLLHKRRRALGLHGKKS-- ------ GKPPLQNNEKE MPVLSRPRP----W-RGNTLKRTAVLLALAAYGAHKVYPLVRQCL-APARGLQAPAGEPTQEASGV------- MTHMLNAAADRVKWTRSSAA CLVA-AAYALKTLYPIIGKRLKQSGHGKKKAAAYPAAENTEILHCTETIKRAA GKKERAVVDKVFFSRLIQILKIMVPRTFCKETGYLVLIAVMLVSRTYCDVWMIQNGTLIESGIIGRSRKDFKR -AAAKAGMNRVFLQRLLWLLRLLFPRVLCRETGLLALHSAALVSRTFLSVYVARLDGRLARCIARKDPRAFGW CEKPSPGVNADFFKQLLELRKILFPKLVTTETGWLCLHSVALISRTFLSIYVAGLDGKIVKSIVEKKPRTFII ++++ ++ ++ + +++++ + + ++ 1 1 1 52 61 73 51 60 72 124 132 145 PMP70 ALDP ALDRP PMP70 ALDP ALDRP TMD1 FIGURE3.BasicaminoacidresiduesintheNH2-terminalregionofPMP70arenotnecessaryforthetargetingofPMP70.A,alignmentoftheNH2-terminal region of human peroxisomal ABC transporters. Login to comment 155 ABCD3 p.Leu71Gln ABCD3 p.Lys120Ala ABCD3 p.Arg124Ala ABCD3 p.Lys53Ala ABCD3 p.Arg117Ala ABCD3 p.Lys61Ala ABCD3 p.Arg77Ala ABCD3 p.Ile70Asn ABCD3 p.Pro76Ala ABCD3 p.Lys28Ala ABCD3 p.Arg119Ala ABCD3 p.Lys38Ala ABCD3 p.Lys123Ala ABCD3 p.Lys54Ala ABCD3 p.Arg29Ala ABCD3 p.Arg31Ala ABCD3 p.Arg66Ala ABCD3 p.Lys72Ala ABCD3 p.Arg30Ala ABCD3 p.Arg56Ala ABCD3 p.Lys39Ala PMP70(AA.1-659 I70N/L71Q) existed in a soluble form in the presence of His-Pex19p and still interacted with His-Pex19p at almost the same level as wild type PMP70 did, but the mutant PMP70 was mislocalized to endoplasmic reticulum-like structures, as (B) (A) PMP70(AA.1-144 K28A/R29A/R30A/R31A/K38A/K39A)-GFP PMP70(AA.1-144 K53A/K54A/R56A/K61A/R66A/K72A/P76A/R77A)-GFP PMP70(AA.1-144 R117A/R119A/K120A/K123A/R124A)-GFP MAAFSKYLT-----------ARNSS-LAGAAFLL--LCLLHKRRRALGLHGKKS-- ----- - GKPPLQNNEKE MPVLSRPRP----W-RGNTLKRTAVLLALAAYGAHKVYPLVRQCL-APARGLQAPAGEPTQEASGV------- MTHMLNAAADRVKWTRSSAA CLVA-AAYALKTLYPIIGKRLKQSGHGKKKAAAYPAAENTEILHCTETI KRAA GKKERAVVDKVFFSRLIQILKIMVPRTFCKETGYLVLIAVMLVSRTYCDVWMIQNGTLIESGIIGRSRKDFKR -AAAKAGMNRVFLQRLLWLLRLLFPRVLCRETGLLALHSAALVSRTFLSVYVARLDGRLARCIARKDPRAFGW CEKPSPGVNADFFKQLLELRKILFPKLVTTETGWLCLHSVALISRTFLSIYVAGLDGKIVKSIVEKKPRTFII ++++ ++ ++ + + + + + + + + ++ 1 1 1 52 61 73 51 60 72 124 132 145 PMP70 ALDP ALDRP PMP70 ALDP ALDRP TMD1 FIGURE3.BasicaminoacidresiduesintheNH2-terminalregionofPMP70arenotnecessaryforthetargetingofPMP70.A,alignmentoftheNH2-terminal region of human peroxisomal ABC transporters. Login to comment 176 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Ala ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Ala ABCD3 p.Leu21Ala ABCD3 p.Leu71Ala ABCD3 p.Leu71Ala ABCD3 p.Leu22Ala Furthermore, only 15% of the fluorescence of PMP70(AA.224-375)- GFP coincided with that of peroxisomes, which was comparable PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP PMP70(AA.1-144 I70N/L71Q)-GFP Not detected Not detected PMP70(AA.1-144 I70A/L71A)-GFP PMP70(AA.1-144 L21A/L22A/L23A)-GFP (A) 45.0 31.0 kDa 45.0 31.0 kDa 01enoN µM MG132 (B) PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP PMP70(AA.1-144 I70N/L71Q)-GFP PMP70(AA.1-659 L21Q/L22Q/L23Q)-GFP PMP70(AA.1-659 I70N/L71Q)-GFP (C) control PMP70(AA.1-144I70N/L71Q)-GFP PMP70(AA.1-144L21Q/L22Q/L23Q)-GFP PMP70(AA.1-144)-GFP control GFP PMP70(AA.1-144I70A/L71A)-GFP PMP70(AA.1-144I70N/L71Q)-GFP PMP70(AA.1-144L21Q/L22Q/L23Q)-GFP PMP70(AA.1-144)-GFP FIGURE 4. Login to comment 177 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Ala ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Ala ABCD3 p.Ile70Ala ABCD3 p.Leu21Ala ABCD3 p.Leu71Ala ABCD3 p.Leu71Ala ABCD3 p.Leu22Ala Furthermore, only 15% of the fluorescence of PMP70(AA.224-375)- GFP coincided with that of peroxisomes, which was comparable PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP PMP70(AA.1-144 I70N/L71Q)-GFP Not detected Not detected PMP70(AA.1-144 I70A/L71A)-GFP PMP70(AA.1-144 L21A/L22A/L23A)-GFP (A) 45.0 31.0 kDa 45.0 31.0 kDa 0 1 e n o N &#b5;M MG132 (B) PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP PMP70(AA.1-144 I70N/L71Q)-GFP PMP70(AA.1-659 L21Q/L22Q/L23Q)-GFP PMP70(AA.1-659 I70N/L71Q)-GFP (C) control PMP70(AA.1-144 I70N/L71Q)-GFP PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP PMP70(AA.1-144)-GFP control GFP PMP70(AA.1-144 I70A/L71A)-GFP PMP70(AA.1-144 I70N/L71Q)-GFP PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP PMP70(AA.1-144)-GFP FIGURE 4. Login to comment 178 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Ala ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Ala ABCD3 p.Leu21Ala ABCD3 p.Leu71Ala ABCD3 p.Leu22Ala A, PMP70(AA.1-144 L21A/L22A/L23A)-GFP, PMP70(AA.1-144 I70A/L71A)-GFP, PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP, and PMP70(AA.1-144 I70N/L71Q)-GFP were expressed in CHO cells. The subcellular distribution of the fusion proteins was compared with the localization of the endogenous PMP70 detected by immunofluorescence staining with anti-PMP70 COOH-terminal antibody. The peroxisomal staining pattern is shown on the left, EGFP fluorescence in the center, and a superimposition of both stains on the right. Login to comment 179 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Ala ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Ala ABCD3 p.Leu21Ala ABCD3 p.Leu71Ala ABCD3 p.Leu22Ala A, PMP70(AA.1-144 L21A/L22A/L23A)-GFP, PMP70(AA.1-144 I70A/L71A)-GFP, PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP, and PMP70(AA.1-144 I70N/L71Q)-GFP were expressed in CHO cells. The subcellular distribution of the fusion proteins was compared with the localization of the endogenous PMP70 detected by immunofluorescence staining with anti-PMP70 COOH-terminal antibody. The peroxisomal staining pattern is shown on the left, EGFP fluorescence in the center, and a superimposition of both stains on the right. Login to comment 181 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn C, PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP, PMP70(AA.1-144 I70N/L71Q)-GFP, PMP70(AA.1-659 L21Q/L22Q/L23Q)-GFP, and PMP70(AA.1-659 I70N/L71Q)-GFP were expressed in CHO cells. Login to comment 182 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn After incubation with 10 ␮M MG132 for 10 h, the subcellular distribution of PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP and PMP70(AA.1-144 I70N/L71Q)-GFP wascomparedwiththelocalizationoftheendogenousPMP70detectedbyimmunofluorescencestainingwith anti-PMP70 COOH-terminal antibody, and the subcellular distribution of PMP70(AA.1-659 L21Q/L22Q/L23Q)- GFP and PMP70(AA.1-659 I70N/L71Q)-GFP was compared with the localization of catalase. Login to comment 183 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn After incubation with 10 òe;M MG132 for 10 h, the subcellular distribution of PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP and PMP70(AA.1-144 I70N/L71Q)-GFP wascomparedwiththelocalizationoftheendogenousPMP70detectedbyimmunofluorescencestainingwith anti-PMP70 COOH-terminal antibody, and the subcellular distribution of PMP70(AA.1-659 L21Q/L22Q/L23Q)- GFP and PMP70(AA.1-659 I70N/L71Q)-GFP was compared with the localization of catalase. Login to comment 191 ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn GFP-PMP70(AA.263-375 I307N/L308Q) was detected only in the presence of MG132. Login to comment 192 ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn GFP-PMP70(AA.263-375 I307N/L308Q) was detected only in the presence of MG132. Login to comment 193 ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn However, under the same conditions, GFP-PMP70(AA.263-375 I307N/L308Q) did not show any peroxisomal localization (Fig. 8). Login to comment 194 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn The mutation also affected the targeting of full-length PMP70, so PMP70(AA.1-659 I307N/L308Q)-GFP was not localized to peroxisomes as PMP70(AA.1-659 L21Q/L22Q/L23Q)-GFP and PMP70(AA.1-659 I70N/L71Q)-GFP were not (see Fig. 4C). Login to comment 195 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn The mutation also affected the targeting of full-length PMP70, so PMP70(AA.1-659 I307N/L308Q)-GFP was not localized to peroxisomes as PMP70(AA.1-659 L21Q/L22Q/L23Q)-GFP and PMP70(AA.1-659 I70N/L71Q)-GFP were not (see Fig. 4C). Login to comment 196 ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn To characterize the hydrophobic motif in the targeting process of PMP70 more precisely, we examined the interaction between PMP70(AA.1-659 I307N/L308Q) and Pex19p. Login to comment 197 ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn As shown in Fig. 9, PMP70(AA.1-659 I307N/L308Q) translated in the wheat germ in vitro translation system was almost recovered as a soluble protein in the presence of purified His-Pex19p, and PMP70(AA.1-659 I307N/L308Q) interacted with Pex19p at almost the same efficiency as PMP70(AA.1-659). Login to comment 198 ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn As shown in Fig. 9, PMP70(AA.1-659 I307N/L308Q) translated in the wheat germ in vitro translation system was almost recovered as a soluble protein in the presence of purified His-Pex19p, and PMP70(AA.1-659 I307N/L308Q) interacted with Pex19p at almost the same efficiency as PMP70(AA.1-659). Login to comment 201 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn However, PMP70(AA.176-276)-GFP as well as GFP-PMP70(AA.176-276), which corresponded to TMD3-TMD4, did not show any peroxisomal localization, excluding the possibility that only two TMDs and a T S P T S P PMP70 I70N/L71Q - + kDa PMP70 L21Q/L22Q/L23QPMP70 66.2 T S P T S P T S P T S P His-Pex19p - + - + (A) PMP70 L21Q/L22Q/L23Q PMP70 I70N/L71QPMP70 0 20 40 60 soluble% (B) kDa PMP70 L21Q/L22Q/L23Q PMP70 I70N/L71Q 10% input ppt PMP70 10% input ppt 10% input ppt 66.2 (C) 0 20 40 60 80 100 120 relativebound% PMP70 L21Q/L22Q/L23Q PMP70 I70N/L71Q PMP70 (D) FIGURE 5. Login to comment 202 ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn ABCD3 p.Ile70Asn However, PMP70(AA.176-276)-GFP as well as GFP-PMP70(AA.176-276), which corresponded to TMD3-TMD4, did not show any peroxisomal localization, excluding the possibility that only two TMDs and a T S P T S P PMP70 I70N/L71Q - + kDa PMP70 L21Q/L22Q/L23Q PMP70 66.2 T S P T S P T S P T S P His-Pex19p - + - + (A) PMP70 L21Q/L22Q/L23Q PMP70 I70N/L71Q PMP70 0 20 40 60 soluble % (B) kDa PMP70 L21Q/L22Q/L23Q PMP70 I70N/L71Q 10% input ppt PMP70 10% input ppt 10% input ppt 66.2 (C) 0 20 40 60 80 100 120 relative bound % PMP70 L21Q/L22Q/L23Q PMP70 I70N/L71Q PMP70 (D) FIGURE 5. Login to comment 236 ABCD3 p.Leu23Ala ABCD3 p.Ile70Ala ABCD3 p.Leu21Ala ABCD3 p.Leu71Ala ABCD3 p.Leu22Ala 2) PMP70(AA.1-144 L21A/L22A/ L23A)-GFP and PMP70(AA.1-144 I70A/L71A)-GFP, which possess a hydrophobic property similar to that of PMP70(AA.1-144)-GFP, were localized to peroxisomes. Login to comment 237 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Ala ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn ABCD3 p.Ile70Ala ABCD3 p.Leu21Ala ABCD3 p.Leu71Ala ABCD3 p.Leu22Ala On the other hand, both PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP and PMP70(AA.1-144 I70N/L71Q)- GFP were degraded by proteasomes and did not show any peroxisomal localization even in the presence of proteasome inhibitor (Fig. 4). Login to comment 238 ABCD3 p.Leu71Gln ABCD3 p.Leu22Gln ABCD3 p.Leu21Gln ABCD3 p.Leu23Gln ABCD3 p.Ile70Asn On the other hand, both PMP70(AA.1-144 L21Q/L22Q/L23Q)-GFP and PMP70(AA.1-144 I70N/L71Q)- GFP were degraded by proteasomes and did not show any peroxisomal localization even in the presence of proteasome inhibitor (Fig. 4). Login to comment 243 ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Leu308Ala ABCD3 p.Ile307Ala Peroxisome Targeting Signal of PMP70 NOVEMBER 16, 2007•VOLUME 282•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 33841 acid pair existing just adjacent to TMD5 was important for the targeting to peroxisomes; GFP-PMP70(AA.263-375 I307A/L308A) was directed to peroxisomes, but GFP-PMP70(AA.263-375 I307N/L308Q) was not (Fig. 8). Login to comment 244 ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Leu308Ala ABCD3 p.Ile307Ala Peroxisome Targeting Signal of PMP70 NOVEMBER 16, 2007ߦVOLUME 282ߦNUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 33841 acid pair existing just adjacent to TMD5 was important for the targeting to peroxisomes; GFP-PMP70(AA.263-375 I307A/L308A) was directed to peroxisomes, but GFP-PMP70(AA.263-375 I307N/L308Q) was not (Fig. 8). Login to comment 257 ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn GFP-PMP70(AA.263-375 I307N/L308Q) PMP70(AA.1-659 I307N/L308Q)-GFP GFP-PMP70(AA.263-375) FIGURE 8. Login to comment 258 ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn GFP-PMP70(AA.263-375 I307N/L308Q) PMP70(AA.1-659 I307N/L308Q)-GFP GFP-PMP70(AA.263-375) FIGURE 8. Login to comment 259 ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn GFP-PMP70(AA.264-375 I307N/L308Q) and PMP70(AA.1-659 I307N/L308Q)-GFP, which are disrupted in the hydrophobic property adjacent to the NH2-terminal side of TMD5, were expressed in CHO cells. Login to comment 260 ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn After incubation with 10 ␮M MG132 for 10 h, the subcellular distribution of GFP-PMP70(AA.264-375 I307N/L308Q) was compared with the localization of the endogenous PMP70 detected by immunofluorescence staining with anti-PMP70 COOH-terminal antibody,andthesubcellulardistributionofPMP70(AA.1-659I307N/L308Q)- GFP was compared with the localization of catalase. Login to comment 261 ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn After incubation with 10 òe;M MG132 for 10 h, the subcellular distribution of GFP-PMP70(AA.264-375 I307N/L308Q) was compared with the localization of the endogenous PMP70 detected by immunofluorescence staining with anti-PMP70 COOH-terminal antibody,andthesubcellulardistributionofPMP70(AA.1-659I307N/L308Q)- GFP was compared with the localization of catalase. Login to comment 262 ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn (B) (A) 0 20 40 60 80 100 120 relativebound% PMP70 I307N/L308Q PMP70 kDa PMP70 I307N/L308Q 10% input ppt PMP70 10% input ppt 66.2 FIGURE 9. Login to comment 263 ABCD3 p.Leu308Gln ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn ABCD3 p.Ile307Asn (B) (A) 0 20 40 60 80 100 120 relative bound % PMP70 I307N/L308Q PMP70 kDa PMP70 I307N/L308Q 10% input ppt PMP70 10% input ppt 66.2 FIGURE 9. Login to comment 264 ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn A, PMP70 and PMP70(I307N/L308Q) were translated and labeled with [35 S]methionine invitrointhepresenceofHis-Pex19p.Aftercentrifugationat17,000ϫgfor20 min at 4 °C, soluble proteins were subjected to immunoprecipitation using anti-His G antibody. Co-purified proteins were analyzed by SDS-PAGE followed by autoradiography. The arrowhead indicates the position of PMP70 and the mutant. Login to comment 265 ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn A, PMP70 and PMP70(I307N/L308Q) were translated and labeled with [35 S]methionine invitrointhepresenceofHis-Pex19p.Aftercentrifugationat17,000afb;gfor20 min at 4 &#b0;C, soluble proteins were subjected to immunoprecipitation using anti-His G antibody. Co-purified proteins were analyzed by SDS-PAGE followed by autoradiography. The arrowhead indicates the position of PMP70 and the mutant. Login to comment 278 ABCD3 p.Leu71Gln ABCD3 p.Ile70Asn ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn However, we also found that PMP70(AA.1-659 I70N/L71Q) and PMP70(AA.1-659 I307N/L308Q) lost the targeting activity, although these mutant proteins still interacted with Pex19p and were solubilized by Pex19p, suggesting that the targeting element and Pex19p-binding site of PMP70 are functionally separated. Login to comment 279 ABCD3 p.Leu71Gln ABCD3 p.Ile70Asn ABCD3 p.Leu308Gln ABCD3 p.Ile307Asn However, we also found that PMP70(AA.1-659 I70N/L71Q) and PMP70(AA.1-659 I307N/L308Q) lost the targeting activity, although these mutant proteins still interacted with Pex19p and were solubilized by Pex19p, suggesting that the targeting element and Pex19p-binding site of PMP70 are functionally separated. Login to comment