Abstract
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.
Highlights
-oxidation of fatty acids, especially very long-chain fatty acids, and synthesis of ether phospholipids (1)
Dyer et al (35) reported that the targeting information of PMP47 resides on the matrix-oriented loop between transmembrane domain 4 (TMD4) and TMD5, which is enriched in positively charged amino acids. membrane protein targeting signal (mPTS) have been determined for several Peroxisomal membrane proteins (PMPs), including Pex3p, PMP22, and PMP34 (36 – 40)
Most of the PMPs are synthesized on free cytosolic polydeleted of the NH2-terminal 20-kDa region adjacent to TMD3 was associated with peroxisomes (45)
Summary
Materials—pEGFP-N1 and pEGFP-C3 were purchased from Clontech (Palo Alto, CA). pQE30 and pEU3-NII were from Qiagen (Valencia, CA) and TOYOBO (Osaka, Japan), respectively. I70N/L71Q)-GFP were not localized to peroxisomes as negative controls (see Fig. 4) These data suggest that these basic clusters are not essential for the targeting of PMP70, and the mPTS of PMP70 is located in another part of the molecule. Hydrophobic Motifs Adjacent to the NH2-terminal Side of TMD1 Are Important for the Stability and the Peroxisomal Targeting of PMP70—Based on the hydropathy profiling, peroxisomal ABC proteins possess two hydrophobic segments adjacent to the NH2-terminal side of TMD1 (Fig. 3A) 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.
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