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Multiple distinct targeting signals in integral peroxisomal membrane proteins.

Jones JM, Morrell JC, Gould SJ - J. Cell Biol. (2001)

Bottom Line: We also show that another integral PMP, the peroxin PEX13, also contains two independent sets of peroxisomal targeting information.These results challenge a major assumption of most PMP targeting studies.In addition, we demonstrate that PEX19, a factor required for peroxisomal membrane biogenesis, interacts with the two minimal targeting regions of PMP34.

View Article: PubMed Central - PubMed

Affiliation: The Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Peroxisomal proteins are synthesized on free polysomes and then transported from the cytoplasm to peroxisomes. This process is mediated by two short well-defined targeting signals in peroxisomal matrix proteins, but a well-defined targeting signal has not yet been described for peroxisomal membrane proteins (PMPs). One assumption in virtually all prior studies of PMP targeting is that a given protein contains one, and only one, distinct targeting signal. Here, we show that the metabolite transporter PMP34, an integral PMP, contains at least two nonoverlapping sets of targeting information, either of which is sufficient for insertion into the peroxisome membrane. We also show that another integral PMP, the peroxin PEX13, also contains two independent sets of peroxisomal targeting information. These results challenge a major assumption of most PMP targeting studies. In addition, we demonstrate that PEX19, a factor required for peroxisomal membrane biogenesis, interacts with the two minimal targeting regions of PMP34. Together, these results raise the interesting possibility that PMP import may require novel mechanisms to ensure the solubility of integral PMPs before their insertion in the peroxisome membrane, and that PEX19 may play a central role in this process.

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Two distinct regions of PMP34 are sufficient for peroxisomal targeting. Human skin fibroblasts expressing PMP34aa1–147/3xmyc (a and b) or PMP34aa244–307/3xmyc (c and d) were processed for double indirect immunofluorescence by fixing cells and permeabilizing with Triton X-100. The distribution of each PMP34 mutant was examined using anti-myc antibodies (a and c) and antibodies to the peroxisomal marker protein PMP70 (b and d). Bar, 15 μm.
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Figure 3: Two distinct regions of PMP34 are sufficient for peroxisomal targeting. Human skin fibroblasts expressing PMP34aa1–147/3xmyc (a and b) or PMP34aa244–307/3xmyc (c and d) were processed for double indirect immunofluorescence by fixing cells and permeabilizing with Triton X-100. The distribution of each PMP34 mutant was examined using anti-myc antibodies (a and c) and antibodies to the peroxisomal marker protein PMP70 (b and d). Bar, 15 μm.

Mentions: We first made a series of deletions starting at the COOH terminus of the protein and found that the NH2-terminal 147 amino acids were capable of targeting to peroxisomes (Fig. 3, a and b). Truncation mutants shorter than this either failed to express in multiple trials or did not target to peroxisomes. Next, we made a series of truncations beginning from the NH2 terminus and found that the COOH-terminal 64 amino acids of PMP34 retained excellent targeting to peroxisomes (Fig. 3c and Fig. d). In these studies, PMP34 truncations that targeted to peroxisomes but no other compartments were designated +++, those that targeted to peroxisomes and targeted to other compartments in some cells were designated ++, those that targeted to peroxisomes and targeted to other compartments in all cells were designated +, and those that showed no peroxisomal targeting were designated −. Most other studies of PMP targeting information have assumed that each PMP contains a single targeting signal (Baerends et al. 1996, Baerends et al. 2000; Wiemer et al. 1996; Kammerer et al. 1998; Pause et al. 2000; Snyder et al. 2000), including some from our own laboratory (Sacksteder et al. 2000). Thus, the identification of two discrete targeting signals in PMP34 was entirely unexpected.


Multiple distinct targeting signals in integral peroxisomal membrane proteins.

Jones JM, Morrell JC, Gould SJ - J. Cell Biol. (2001)

Two distinct regions of PMP34 are sufficient for peroxisomal targeting. Human skin fibroblasts expressing PMP34aa1–147/3xmyc (a and b) or PMP34aa244–307/3xmyc (c and d) were processed for double indirect immunofluorescence by fixing cells and permeabilizing with Triton X-100. The distribution of each PMP34 mutant was examined using anti-myc antibodies (a and c) and antibodies to the peroxisomal marker protein PMP70 (b and d). Bar, 15 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192020&req=5

Figure 3: Two distinct regions of PMP34 are sufficient for peroxisomal targeting. Human skin fibroblasts expressing PMP34aa1–147/3xmyc (a and b) or PMP34aa244–307/3xmyc (c and d) were processed for double indirect immunofluorescence by fixing cells and permeabilizing with Triton X-100. The distribution of each PMP34 mutant was examined using anti-myc antibodies (a and c) and antibodies to the peroxisomal marker protein PMP70 (b and d). Bar, 15 μm.
Mentions: We first made a series of deletions starting at the COOH terminus of the protein and found that the NH2-terminal 147 amino acids were capable of targeting to peroxisomes (Fig. 3, a and b). Truncation mutants shorter than this either failed to express in multiple trials or did not target to peroxisomes. Next, we made a series of truncations beginning from the NH2 terminus and found that the COOH-terminal 64 amino acids of PMP34 retained excellent targeting to peroxisomes (Fig. 3c and Fig. d). In these studies, PMP34 truncations that targeted to peroxisomes but no other compartments were designated +++, those that targeted to peroxisomes and targeted to other compartments in some cells were designated ++, those that targeted to peroxisomes and targeted to other compartments in all cells were designated +, and those that showed no peroxisomal targeting were designated −. Most other studies of PMP targeting information have assumed that each PMP contains a single targeting signal (Baerends et al. 1996, Baerends et al. 2000; Wiemer et al. 1996; Kammerer et al. 1998; Pause et al. 2000; Snyder et al. 2000), including some from our own laboratory (Sacksteder et al. 2000). Thus, the identification of two discrete targeting signals in PMP34 was entirely unexpected.

Bottom Line: We also show that another integral PMP, the peroxin PEX13, also contains two independent sets of peroxisomal targeting information.These results challenge a major assumption of most PMP targeting studies.In addition, we demonstrate that PEX19, a factor required for peroxisomal membrane biogenesis, interacts with the two minimal targeting regions of PMP34.

View Article: PubMed Central - PubMed

Affiliation: The Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Peroxisomal proteins are synthesized on free polysomes and then transported from the cytoplasm to peroxisomes. This process is mediated by two short well-defined targeting signals in peroxisomal matrix proteins, but a well-defined targeting signal has not yet been described for peroxisomal membrane proteins (PMPs). One assumption in virtually all prior studies of PMP targeting is that a given protein contains one, and only one, distinct targeting signal. Here, we show that the metabolite transporter PMP34, an integral PMP, contains at least two nonoverlapping sets of targeting information, either of which is sufficient for insertion into the peroxisome membrane. We also show that another integral PMP, the peroxin PEX13, also contains two independent sets of peroxisomal targeting information. These results challenge a major assumption of most PMP targeting studies. In addition, we demonstrate that PEX19, a factor required for peroxisomal membrane biogenesis, interacts with the two minimal targeting regions of PMP34. Together, these results raise the interesting possibility that PMP import may require novel mechanisms to ensure the solubility of integral PMPs before their insertion in the peroxisome membrane, and that PEX19 may play a central role in this process.

Show MeSH
Related in: MedlinePlus