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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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PMP34myc localizes to peroxisomes by a pathway distinct from that used by matrix proteins. (a) Lysates of mock-transfected human fibroblasts, fibroblasts expressing PMP34myc, and PEX10-deficient PBD100 fibroblasts expressing PMP34myc were analyzed by immunoblot using anti-myc and anticatalase antibodies. (b–e) Human skin fibroblasts expressing PMP34myc were processed for double indirect immunofluorescence by fixing cells and permeabilizing them with 1% Triton X-100. The distribution of PMP34myc was examined using anti-myc antibodies (b) and anti-PMP70 antibodies (c). The distribution of PMP34myc was also examined in fibroblasts of the PEX10-deficient cell line PBD100, again by double indirect immunofluorescence using anti-myc antibodies (d) and anti-PMP70 antibodies (e). Bar, 15 μm.
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Figure 1: PMP34myc localizes to peroxisomes by a pathway distinct from that used by matrix proteins. (a) Lysates of mock-transfected human fibroblasts, fibroblasts expressing PMP34myc, and PEX10-deficient PBD100 fibroblasts expressing PMP34myc were analyzed by immunoblot using anti-myc and anticatalase antibodies. (b–e) Human skin fibroblasts expressing PMP34myc were processed for double indirect immunofluorescence by fixing cells and permeabilizing them with 1% Triton X-100. The distribution of PMP34myc was examined using anti-myc antibodies (b) and anti-PMP70 antibodies (c). The distribution of PMP34myc was also examined in fibroblasts of the PEX10-deficient cell line PBD100, again by double indirect immunofluorescence using anti-myc antibodies (d) and anti-PMP70 antibodies (e). Bar, 15 μm.

Mentions: To distinguish between endogenously expressed PMP34 and PMP34 proteins expressed from plasmids, the plasmid-encoded proteins all contained the c-myc epitope at their COOH terminus. The full-length PMP34myc fusion protein was expressed in wild-type human fibroblasts (Fig. 1 a) and was efficiently targeted to peroxisomes (b and c). The Zellweger syndrome fibroblast cell line PBD100 is unable to import peroxisomal matrix proteins but imports PMPs normally (Warren et al. 1998). PMP34myc was expressed in PBD100 cells (Fig. 1 a) and was targeted to peroxisomes (d and e), demonstrating that PMP34myc is targeted to peroxisomes by a membrane protein targeting pathway, as previously suggested (Wylin et al. 1999).


Multiple distinct targeting signals in integral peroxisomal membrane proteins.

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

PMP34myc localizes to peroxisomes by a pathway distinct from that used by matrix proteins. (a) Lysates of mock-transfected human fibroblasts, fibroblasts expressing PMP34myc, and PEX10-deficient PBD100 fibroblasts expressing PMP34myc were analyzed by immunoblot using anti-myc and anticatalase antibodies. (b–e) Human skin fibroblasts expressing PMP34myc were processed for double indirect immunofluorescence by fixing cells and permeabilizing them with 1% Triton X-100. The distribution of PMP34myc was examined using anti-myc antibodies (b) and anti-PMP70 antibodies (c). The distribution of PMP34myc was also examined in fibroblasts of the PEX10-deficient cell line PBD100, again by double indirect immunofluorescence using anti-myc antibodies (d) and anti-PMP70 antibodies (e). Bar, 15 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192020&req=5

Figure 1: PMP34myc localizes to peroxisomes by a pathway distinct from that used by matrix proteins. (a) Lysates of mock-transfected human fibroblasts, fibroblasts expressing PMP34myc, and PEX10-deficient PBD100 fibroblasts expressing PMP34myc were analyzed by immunoblot using anti-myc and anticatalase antibodies. (b–e) Human skin fibroblasts expressing PMP34myc were processed for double indirect immunofluorescence by fixing cells and permeabilizing them with 1% Triton X-100. The distribution of PMP34myc was examined using anti-myc antibodies (b) and anti-PMP70 antibodies (c). The distribution of PMP34myc was also examined in fibroblasts of the PEX10-deficient cell line PBD100, again by double indirect immunofluorescence using anti-myc antibodies (d) and anti-PMP70 antibodies (e). Bar, 15 μm.
Mentions: To distinguish between endogenously expressed PMP34 and PMP34 proteins expressed from plasmids, the plasmid-encoded proteins all contained the c-myc epitope at their COOH terminus. The full-length PMP34myc fusion protein was expressed in wild-type human fibroblasts (Fig. 1 a) and was efficiently targeted to peroxisomes (b and c). The Zellweger syndrome fibroblast cell line PBD100 is unable to import peroxisomal matrix proteins but imports PMPs normally (Warren et al. 1998). PMP34myc was expressed in PBD100 cells (Fig. 1 a) and was targeted to peroxisomes (d and e), demonstrating that PMP34myc is targeted to peroxisomes by a membrane protein targeting pathway, as previously suggested (Wylin et al. 1999).

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