ABCMdb

ABCD3 p.Ile70Asn

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Publications

PMID: 17761678 [PubMed] Kashiwayama Y et al: "Hydrophobic regions adjacent to transmembrane domains 1 and 5 are important for the targeting of the 70-kDa peroxisomal membrane protein."

No. Sentence Comment

88 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
129 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
135 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 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 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 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 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 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 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
154 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
176 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
178 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 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 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
194 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
201 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
237 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
278 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
89 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
130 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
155 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
177 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
179 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
183 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
195 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
202 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
238 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
279 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