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PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins.

Jones JM, Morrell JC, Gould SJ - J. Cell Biol. (2004)

Bottom Line: Here, we demonstrate that PEX19 binds and stabilizes newly synthesized PMPs in the cytosol, binds to multiple PMP targeting signals (mPTSs), interacts with the hydrophobic domains of PMP targeting signals, and is essential for PMP targeting and import.These results show that PEX19 functions as both a chaperone and an import receptor for newly synthesized PMPs.We also demonstrate the existence of two PMP import mechanisms and two classes of mPTSs: class 1 mPTSs, which are bound by PEX19 and imported in a PEX19-dependent manner, and class 2 mPTSs, which are not bound by PEX19 and mediate protein import independently of PEX19.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

ABSTRACT
Integral peroxisomal membrane proteins (PMPs) are synthesized in the cytoplasm and imported posttranslationally. Here, we demonstrate that PEX19 binds and stabilizes newly synthesized PMPs in the cytosol, binds to multiple PMP targeting signals (mPTSs), interacts with the hydrophobic domains of PMP targeting signals, and is essential for PMP targeting and import. These results show that PEX19 functions as both a chaperone and an import receptor for newly synthesized PMPs. We also demonstrate the existence of two PMP import mechanisms and two classes of mPTSs: class 1 mPTSs, which are bound by PEX19 and imported in a PEX19-dependent manner, and class 2 mPTSs, which are not bound by PEX19 and mediate protein import independently of PEX19.

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Peroxisomes persist for multiple days after inhibition of PEX19. (A) Treatment with PEX19 and PEX5 siRNA mediates specific reductions in PEX19 and PEX5 levels. Wild-type human fibroblasts were subjected to electroporation with PEX19 siRNA (top) or PEX5 siRNA (bottom). Equal amounts of protein from daily cell samples after siRNA treatment were separated by SDS-PAGE and processed for immunoblot using antibodies to PEX19, PEX5, and PEX13. (B) Peroxisomes persist for >3 d despite inhibition of PEX19. Daily cell samples after treatment with PEX19 siRNA (top) or PEX5 siRNA (bottom) were processed for indirect immunofluorescence using antibodies to the peroxisomal membrane marker PMP70. Bar, 15 μM.
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fig4: Peroxisomes persist for multiple days after inhibition of PEX19. (A) Treatment with PEX19 and PEX5 siRNA mediates specific reductions in PEX19 and PEX5 levels. Wild-type human fibroblasts were subjected to electroporation with PEX19 siRNA (top) or PEX5 siRNA (bottom). Equal amounts of protein from daily cell samples after siRNA treatment were separated by SDS-PAGE and processed for immunoblot using antibodies to PEX19, PEX5, and PEX13. (B) Peroxisomes persist for >3 d despite inhibition of PEX19. Daily cell samples after treatment with PEX19 siRNA (top) or PEX5 siRNA (bottom) were processed for indirect immunofluorescence using antibodies to the peroxisomal membrane marker PMP70. Bar, 15 μM.

Mentions: The data presented thus far support the hypothesis that PEX19 is a chaperone and import receptor for newly synthesized PMPs. However, this hypothesis demands that PEX19 play a direct role in PMP import. Although the absence of peroxisomes in PEX19-deficient cells is consistent with this hypothesis, this phenotype could also reflect a role for PEX19 in peroxisome division, lipid import, or peroxisome synthesis de novo. To determine whether PEX19 plays a direct role in PMP import, we used RNA interference (RNAi) to elicit a transient inhibition of PEX19 in normal human fibroblasts (Elbashir et al., 2001). A 21-bp siRNA duplex corresponding to a unique sequence of human PEX19 mRNA was transfected into 5756-TI cells and levels of PEX19 protein were followed over a span of 5 d. PEX19 protein levels were severely reduced by day two, remained low on days three and four, and rose slightly by day five (Fig. 4 A). In contrast, the levels of two other peroxins, the import receptor for peroxisomal matrix proteins, PEX5, and the integral PMP, PEX13, were relatively unaffected. Transfection with siRNA specific to PEX5 resulted in a reduction in PEX5 protein levels, with little or no change in PEX19 or PEX13 abundance. Immunofluorescence studies of cells treated with PEX5 and PEX19 siRNAs showed numerous peroxisomes over the first 3 d (Fig. 4 B), demonstrating that PEX19 siRNA could be used to inhibit PEX19 in cells that still contained peroxisomes. Prolonged treatment with PEX19-specific siRNA caused a reduction in peroxisome number and the loss of peroxisomes from some cells (unpublished data). This finding is consistent with the known phenotype of PEX19- cells, which lack peroxisomes.


PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins.

Jones JM, Morrell JC, Gould SJ - J. Cell Biol. (2004)

Peroxisomes persist for multiple days after inhibition of PEX19. (A) Treatment with PEX19 and PEX5 siRNA mediates specific reductions in PEX19 and PEX5 levels. Wild-type human fibroblasts were subjected to electroporation with PEX19 siRNA (top) or PEX5 siRNA (bottom). Equal amounts of protein from daily cell samples after siRNA treatment were separated by SDS-PAGE and processed for immunoblot using antibodies to PEX19, PEX5, and PEX13. (B) Peroxisomes persist for >3 d despite inhibition of PEX19. Daily cell samples after treatment with PEX19 siRNA (top) or PEX5 siRNA (bottom) were processed for indirect immunofluorescence using antibodies to the peroxisomal membrane marker PMP70. Bar, 15 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171958&req=5

fig4: Peroxisomes persist for multiple days after inhibition of PEX19. (A) Treatment with PEX19 and PEX5 siRNA mediates specific reductions in PEX19 and PEX5 levels. Wild-type human fibroblasts were subjected to electroporation with PEX19 siRNA (top) or PEX5 siRNA (bottom). Equal amounts of protein from daily cell samples after siRNA treatment were separated by SDS-PAGE and processed for immunoblot using antibodies to PEX19, PEX5, and PEX13. (B) Peroxisomes persist for >3 d despite inhibition of PEX19. Daily cell samples after treatment with PEX19 siRNA (top) or PEX5 siRNA (bottom) were processed for indirect immunofluorescence using antibodies to the peroxisomal membrane marker PMP70. Bar, 15 μM.
Mentions: The data presented thus far support the hypothesis that PEX19 is a chaperone and import receptor for newly synthesized PMPs. However, this hypothesis demands that PEX19 play a direct role in PMP import. Although the absence of peroxisomes in PEX19-deficient cells is consistent with this hypothesis, this phenotype could also reflect a role for PEX19 in peroxisome division, lipid import, or peroxisome synthesis de novo. To determine whether PEX19 plays a direct role in PMP import, we used RNA interference (RNAi) to elicit a transient inhibition of PEX19 in normal human fibroblasts (Elbashir et al., 2001). A 21-bp siRNA duplex corresponding to a unique sequence of human PEX19 mRNA was transfected into 5756-TI cells and levels of PEX19 protein were followed over a span of 5 d. PEX19 protein levels were severely reduced by day two, remained low on days three and four, and rose slightly by day five (Fig. 4 A). In contrast, the levels of two other peroxins, the import receptor for peroxisomal matrix proteins, PEX5, and the integral PMP, PEX13, were relatively unaffected. Transfection with siRNA specific to PEX5 resulted in a reduction in PEX5 protein levels, with little or no change in PEX19 or PEX13 abundance. Immunofluorescence studies of cells treated with PEX5 and PEX19 siRNAs showed numerous peroxisomes over the first 3 d (Fig. 4 B), demonstrating that PEX19 siRNA could be used to inhibit PEX19 in cells that still contained peroxisomes. Prolonged treatment with PEX19-specific siRNA caused a reduction in peroxisome number and the loss of peroxisomes from some cells (unpublished data). This finding is consistent with the known phenotype of PEX19- cells, which lack peroxisomes.

Bottom Line: Here, we demonstrate that PEX19 binds and stabilizes newly synthesized PMPs in the cytosol, binds to multiple PMP targeting signals (mPTSs), interacts with the hydrophobic domains of PMP targeting signals, and is essential for PMP targeting and import.These results show that PEX19 functions as both a chaperone and an import receptor for newly synthesized PMPs.We also demonstrate the existence of two PMP import mechanisms and two classes of mPTSs: class 1 mPTSs, which are bound by PEX19 and imported in a PEX19-dependent manner, and class 2 mPTSs, which are not bound by PEX19 and mediate protein import independently of PEX19.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

ABSTRACT
Integral peroxisomal membrane proteins (PMPs) are synthesized in the cytoplasm and imported posttranslationally. Here, we demonstrate that PEX19 binds and stabilizes newly synthesized PMPs in the cytosol, binds to multiple PMP targeting signals (mPTSs), interacts with the hydrophobic domains of PMP targeting signals, and is essential for PMP targeting and import. These results show that PEX19 functions as both a chaperone and an import receptor for newly synthesized PMPs. We also demonstrate the existence of two PMP import mechanisms and two classes of mPTSs: class 1 mPTSs, which are bound by PEX19 and imported in a PEX19-dependent manner, and class 2 mPTSs, which are not bound by PEX19 and mediate protein import independently of PEX19.

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