ABCD3 p.Leu71Ala
| Predicted by SNAP2: | A: N (53%), C: N (66%), D: D (85%), E: D (80%), F: N (78%), G: D (71%), H: D (75%), I: N (82%), K: D (80%), M: N (72%), N: D (75%), P: D (85%), Q: D (71%), R: D (71%), S: D (71%), T: D (66%), V: N (78%), W: N (53%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: N, |
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[hide] Hydrophobic regions adjacent to transmembrane doma... J Biol Chem. 2007 Nov 16;282(46):33831-44. Epub 2007 Aug 30. 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., [PMID:17761678]
Abstract [show]
The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH(2)-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH(2)-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH(2)-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile(307)/Leu(308)) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.
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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.
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ABCD3 p.Leu71Ala 17761678:88:823
status: NEW136 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).
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ABCD3 p.Leu71Ala 17761678:136:215
status: NEW137 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.
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ABCD3 p.Leu71Ala 17761678:137:215
status: NEW176 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.
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ABCD3 p.Leu71Ala 17761678:176:236
status: NEWX
ABCD3 p.Leu71Ala 17761678:176:594
status: NEW178 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.
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ABCD3 p.Leu71Ala 17761678:178:59
status: NEW236 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.
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ABCD3 p.Leu71Ala 17761678:236:63
status: NEW237 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).
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ABCD3 p.Leu71Ala 17761678:237:63
status: NEW89 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.
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ABCD3 p.Leu71Ala 17761678:89:823
status: NEW177 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.
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ABCD3 p.Leu71Ala 17761678:177:236
status: NEWX
ABCD3 p.Leu71Ala 17761678:177:601
status: NEW179 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.
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ABCD3 p.Leu71Ala 17761678:179:59
status: NEW