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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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Related in: MedlinePlus

The two minimal targeting regions of PMP34 are inserted into the peroxisomal membrane. Human skin fibroblasts expressing PMP34aa1–147/3xmyc or PMP34aa244–307/3xmyc were lysed by thorough mixing in hypotonic buffer. Membranes were pelleted by centrifugation, separated from the supernatant, and incubated in alkaline sodium phosphate buffer. Membranes were then pelleted again, separated from the supernatant, and resuspended. All fractions were diluted to the initial volume of whole lysate. Equal volumes of each fraction were then assayed by immunoblot using antibodies against the c-myc epitope, the soluble peroxisomal matrix enzyme catalase, and the integral PMP PEX13.
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Figure 4: The two minimal targeting regions of PMP34 are inserted into the peroxisomal membrane. Human skin fibroblasts expressing PMP34aa1–147/3xmyc or PMP34aa244–307/3xmyc were lysed by thorough mixing in hypotonic buffer. Membranes were pelleted by centrifugation, separated from the supernatant, and incubated in alkaline sodium phosphate buffer. Membranes were then pelleted again, separated from the supernatant, and resuspended. All fractions were diluted to the initial volume of whole lysate. Equal volumes of each fraction were then assayed by immunoblot using antibodies against the c-myc epitope, the soluble peroxisomal matrix enzyme catalase, and the integral PMP PEX13.

Mentions: Peroxisomal localization of the two PMP34 fragments could reflect insertion into the peroxisome membrane. However, it could also have been caused by interaction with a binding partner at the peroxisome surface or by cryptic import into the peroxisome matrix. To address these concerns, fibroblasts expressing the NH2- and COOH-terminal targeting elements of PMP34 were lysed in hypotonic buffer. Soluble proteins were separated from membranes by centrifugation, the membranes were extracted with alkaline sodium carbonate, and the resulting suspension was separated into a membrane pellet and a soluble supernatant. These fractions were then assayed by immunoblot. Proteins containing either the NH2-terminal PTS (PMP34aa1-147/3xmyc) or the COOH-terminal PTS (PMP34aa244-307/3xmyc) remained in the membrane even after carbonate extraction, demonstrating that these signals direct proteins into the peroxisome membrane (Fig. 4). As expected, control immunoblots showed that the peroxisomal matrix enzyme catalase was released to the supernatant by hypotonic lysis and that the integral PMP PEX13 was resistant to carbonate extraction.


Multiple distinct targeting signals in integral peroxisomal membrane proteins.

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

The two minimal targeting regions of PMP34 are inserted into the peroxisomal membrane. Human skin fibroblasts expressing PMP34aa1–147/3xmyc or PMP34aa244–307/3xmyc were lysed by thorough mixing in hypotonic buffer. Membranes were pelleted by centrifugation, separated from the supernatant, and incubated in alkaline sodium phosphate buffer. Membranes were then pelleted again, separated from the supernatant, and resuspended. All fractions were diluted to the initial volume of whole lysate. Equal volumes of each fraction were then assayed by immunoblot using antibodies against the c-myc epitope, the soluble peroxisomal matrix enzyme catalase, and the integral PMP PEX13.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: The two minimal targeting regions of PMP34 are inserted into the peroxisomal membrane. Human skin fibroblasts expressing PMP34aa1–147/3xmyc or PMP34aa244–307/3xmyc were lysed by thorough mixing in hypotonic buffer. Membranes were pelleted by centrifugation, separated from the supernatant, and incubated in alkaline sodium phosphate buffer. Membranes were then pelleted again, separated from the supernatant, and resuspended. All fractions were diluted to the initial volume of whole lysate. Equal volumes of each fraction were then assayed by immunoblot using antibodies against the c-myc epitope, the soluble peroxisomal matrix enzyme catalase, and the integral PMP PEX13.
Mentions: Peroxisomal localization of the two PMP34 fragments could reflect insertion into the peroxisome membrane. However, it could also have been caused by interaction with a binding partner at the peroxisome surface or by cryptic import into the peroxisome matrix. To address these concerns, fibroblasts expressing the NH2- and COOH-terminal targeting elements of PMP34 were lysed in hypotonic buffer. Soluble proteins were separated from membranes by centrifugation, the membranes were extracted with alkaline sodium carbonate, and the resulting suspension was separated into a membrane pellet and a soluble supernatant. These fractions were then assayed by immunoblot. Proteins containing either the NH2-terminal PTS (PMP34aa1-147/3xmyc) or the COOH-terminal PTS (PMP34aa244-307/3xmyc) remained in the membrane even after carbonate extraction, demonstrating that these signals direct proteins into the peroxisome membrane (Fig. 4). As expected, control immunoblots showed that the peroxisomal matrix enzyme catalase was released to the supernatant by hypotonic lysis and that the integral PMP PEX13 was resistant to carbonate extraction.

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