ABCMdb

ABCD3 p.Leu308Gln

Predicted by SNAP2:	A: N (87%), C: N (93%), D: N (72%), E: N (87%), F: N (97%), G: N (82%), H: N (93%), I: N (93%), K: N (82%), M: N (97%), N: N (87%), P: N (72%), Q: N (93%), R: N (82%), S: N (93%), T: N (93%), V: N (97%), W: N (82%), Y: N (93%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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Publications
[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. Login to comment
191 GFP-PMP70(AA.263-375 I307N/L308Q) was detected only in the presence of MG132. Login to comment
193 However, under the same conditions, GFP-PMP70(AA.263-375 I307N/L308Q) did not show any peroxisomal localization (Fig. 8). 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
196 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 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
243 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
257 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 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 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
262 (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
264 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
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
192 GFP-PMP70(AA.263-375 I307N/L308Q) was detected only in the presence of MG132. 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
198 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
244 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
258 GFP-PMP70(AA.263-375 I307N/L308Q) PMP70(AA.1-659 I307N/L308Q)-GFP GFP-PMP70(AA.263-375) FIGURE 8. Login to comment
261 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
263 (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
265 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
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