ABCD3 p.Arg56Ala
| Predicted by SNAP2: | A: D (59%), C: D (59%), D: D (80%), E: D (66%), F: D (66%), G: D (71%), H: N (82%), I: D (53%), K: N (97%), L: D (53%), M: D (53%), N: D (53%), P: D (75%), Q: N (57%), S: N (66%), T: D (53%), V: D (59%), W: D (75%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, S: D, T: D, V: D, W: D, Y: D, |
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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.Arg56Ala 17761678:88:505
status: NEW128 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.
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ABCD3 p.Arg56Ala 17761678:128:96
status: NEW129 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).
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ABCD3 p.Arg56Ala 17761678:129:96
status: NEW154 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.
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ABCD3 p.Arg56Ala 17761678:154:334
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.
X
ABCD3 p.Arg56Ala 17761678:89:505
status: NEW155 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.
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ABCD3 p.Arg56Ala 17761678:155:334
status: NEW