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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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Inhibition of PEX19 causes a specific PMP import defect. (A) Inhibition of PEX19 disrupts import of PMP34 but not import of the peroxisomal matrix protein, PTE1. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA (top), PEX19 siRNA (middle), or PEX5 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and PMP34myc, and processed for indirect immunofluorescence with antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells showing peroxisomal import were counted and scored for import of HA-PTE1 or PMP34myc (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 314; PEX19 siRNA n = 303; PEX5 siRNA n = 67. Bar, 15 μM. (B) Inhibition of PEX19 disrupts mPTS targeting. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA or PEX19 siRNA followed by transfection with plasmids designed to express HA-PTE1 and either PMP34aa244-307/3xmyc (top) or PEX16aa221-336/3xmyc (bottom). Cells were processed for immunofluorescence using anti-HA (left) or anti-myc (middle) antibodies. Cells that imported HA-PTE1 into peroxisomes were scored as to whether PMP34aa244-307/3xmyc or PEX16aa221-336/3xmyc was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. PMP34aa244-307/3xmyc: TRIP8b siRNA n = 306, PEX19 siRNA n = 305; PEX16aa221-336/3xmyc: TRIP8b siRNA n = 301, PEX19 siRNA n = 306. Bar, 15 μM.
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fig5: Inhibition of PEX19 causes a specific PMP import defect. (A) Inhibition of PEX19 disrupts import of PMP34 but not import of the peroxisomal matrix protein, PTE1. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA (top), PEX19 siRNA (middle), or PEX5 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and PMP34myc, and processed for indirect immunofluorescence with antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells showing peroxisomal import were counted and scored for import of HA-PTE1 or PMP34myc (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 314; PEX19 siRNA n = 303; PEX5 siRNA n = 67. Bar, 15 μM. (B) Inhibition of PEX19 disrupts mPTS targeting. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA or PEX19 siRNA followed by transfection with plasmids designed to express HA-PTE1 and either PMP34aa244-307/3xmyc (top) or PEX16aa221-336/3xmyc (bottom). Cells were processed for immunofluorescence using anti-HA (left) or anti-myc (middle) antibodies. Cells that imported HA-PTE1 into peroxisomes were scored as to whether PMP34aa244-307/3xmyc or PEX16aa221-336/3xmyc was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. PMP34aa244-307/3xmyc: TRIP8b siRNA n = 306, PEX19 siRNA n = 305; PEX16aa221-336/3xmyc: TRIP8b siRNA n = 301, PEX19 siRNA n = 306. Bar, 15 μM.

Mentions: Wild-type cells were treated with siRNAs specific for PEX19, PEX5, or a control gene, TRIP8b (Chen et al., 2001). 60 h later, the cells were transfected with a pair of plasmids that express a PMP, PMP34myc, and a peroxisomal matrix protein, HA-PTE1. 100 min after transfection, the cells were fixed and processed for immunofluorescence to assess the import of these marker proteins. To ensure that intact, functioning peroxisomes were present, we examined only those cells that imported either PMP34myc or HA-PTE1 (Fig. 5 A). Under this scheme the majority (75%) of TRIP8b-treated cells showed import of both HA-PTE1 and PMP34myc. As expected from PEX5's well-established role in peroxisomal matrix protein import, inhibition of PEX5 induced a selective deficit in peroxisomal matrix protein import, with 88% of cells importing only PMP34myc and only 12% importing HA-PTE1. The fact that some cells imported HA-PTE1 is consistent with the fact that our electroporation protocol used for siRNA delivery is less than 100% efficient (Chang et al., 1997). Inhibition of PEX19 induced the converse phenotype, a selective defect in PMP import. In PEX19 siRNA-treated cells, the majority of cells (76%) imported only HA-PTE1. Interestingly, rather than being distributed throughout the cytosol, like HA-PTE1 in PEX5 siRNA-treated cells, PMP34myc was simply not seen in the majority of PEX19 siRNA-treated cells. This was likely due to degradation of PMP34myc and is consistent with both the well-documented instability of PMPs in the absence of PEX19 (Hettema et al., 2000; Sacksteder et al., 2000) and the stabilizing effect of PEX19 on PMP34 observed in Fig. 1 G.


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)

Inhibition of PEX19 causes a specific PMP import defect. (A) Inhibition of PEX19 disrupts import of PMP34 but not import of the peroxisomal matrix protein, PTE1. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA (top), PEX19 siRNA (middle), or PEX5 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and PMP34myc, and processed for indirect immunofluorescence with antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells showing peroxisomal import were counted and scored for import of HA-PTE1 or PMP34myc (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 314; PEX19 siRNA n = 303; PEX5 siRNA n = 67. Bar, 15 μM. (B) Inhibition of PEX19 disrupts mPTS targeting. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA or PEX19 siRNA followed by transfection with plasmids designed to express HA-PTE1 and either PMP34aa244-307/3xmyc (top) or PEX16aa221-336/3xmyc (bottom). Cells were processed for immunofluorescence using anti-HA (left) or anti-myc (middle) antibodies. Cells that imported HA-PTE1 into peroxisomes were scored as to whether PMP34aa244-307/3xmyc or PEX16aa221-336/3xmyc was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. PMP34aa244-307/3xmyc: TRIP8b siRNA n = 306, PEX19 siRNA n = 305; PEX16aa221-336/3xmyc: TRIP8b siRNA n = 301, PEX19 siRNA n = 306. Bar, 15 μM.
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fig5: Inhibition of PEX19 causes a specific PMP import defect. (A) Inhibition of PEX19 disrupts import of PMP34 but not import of the peroxisomal matrix protein, PTE1. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA (top), PEX19 siRNA (middle), or PEX5 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and PMP34myc, and processed for indirect immunofluorescence with antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells showing peroxisomal import were counted and scored for import of HA-PTE1 or PMP34myc (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 314; PEX19 siRNA n = 303; PEX5 siRNA n = 67. Bar, 15 μM. (B) Inhibition of PEX19 disrupts mPTS targeting. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA or PEX19 siRNA followed by transfection with plasmids designed to express HA-PTE1 and either PMP34aa244-307/3xmyc (top) or PEX16aa221-336/3xmyc (bottom). Cells were processed for immunofluorescence using anti-HA (left) or anti-myc (middle) antibodies. Cells that imported HA-PTE1 into peroxisomes were scored as to whether PMP34aa244-307/3xmyc or PEX16aa221-336/3xmyc was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. PMP34aa244-307/3xmyc: TRIP8b siRNA n = 306, PEX19 siRNA n = 305; PEX16aa221-336/3xmyc: TRIP8b siRNA n = 301, PEX19 siRNA n = 306. Bar, 15 μM.
Mentions: Wild-type cells were treated with siRNAs specific for PEX19, PEX5, or a control gene, TRIP8b (Chen et al., 2001). 60 h later, the cells were transfected with a pair of plasmids that express a PMP, PMP34myc, and a peroxisomal matrix protein, HA-PTE1. 100 min after transfection, the cells were fixed and processed for immunofluorescence to assess the import of these marker proteins. To ensure that intact, functioning peroxisomes were present, we examined only those cells that imported either PMP34myc or HA-PTE1 (Fig. 5 A). Under this scheme the majority (75%) of TRIP8b-treated cells showed import of both HA-PTE1 and PMP34myc. As expected from PEX5's well-established role in peroxisomal matrix protein import, inhibition of PEX5 induced a selective deficit in peroxisomal matrix protein import, with 88% of cells importing only PMP34myc and only 12% importing HA-PTE1. The fact that some cells imported HA-PTE1 is consistent with the fact that our electroporation protocol used for siRNA delivery is less than 100% efficient (Chang et al., 1997). Inhibition of PEX19 induced the converse phenotype, a selective defect in PMP import. In PEX19 siRNA-treated cells, the majority of cells (76%) imported only HA-PTE1. Interestingly, rather than being distributed throughout the cytosol, like HA-PTE1 in PEX5 siRNA-treated cells, PMP34myc was simply not seen in the majority of PEX19 siRNA-treated cells. This was likely due to degradation of PMP34myc and is consistent with both the well-documented instability of PMPs in the absence of PEX19 (Hettema et al., 2000; Sacksteder et al., 2000) and the stabilizing effect of PEX19 on PMP34 observed in Fig. 1 G.

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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