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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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The mPTS of PEX3 functions via a PEX19-independent pathway. (A) The first 50 aa of PEX3 are sufficient for peroxisomal targeting. Wild-type human fibroblasts were transfected with a plasmid designed to express the PEX3 mPTS (PEX3aa1-50/6xmet3xmyc) and cells were processed for immunofluorescence using antibodies to the c-myc epitope (left) or PMP70 (right). Bar, 15 μM. (B) The mPTS of PEX3 does not coprecipitate with PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either 3xHA-PEX19 (+3xHA-PEX19) or PEX19 (+PEX19). Equal total protein from a membrane-free lysate of each cell sample was subjected to immunoprecipitation with antibodies to the HA epitope. IPs (top) and whole cell lysates (bottom) were processed for immunoblot using anti-myc antibodies. (C) The subcellular distribution of the PEX3 mPTS is not affected by PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either PEX19 (top) or 3xNLS-PEX19 (bottom). Cells were processed for indirect immunofluorescence using anti-myc (left) or anti-PEX19 (right) antibodies. Bar, 15 μM. (D) Inhibition of PEX19 does not affect peroxisomal targeting of the PEX3 mPTS. Wild-type human fibroblasts were subjected to electroporation with either TRIP8b siRNA (top) or PEX19 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and the PEX3 mPTS. Cells were processed for immunofluorescence using antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells importing HA-PTE1 were scored as to whether the PEX3 mPTS was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 311; PEX19 siRNA n = 294. Bar, 15 μM.
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fig6: The mPTS of PEX3 functions via a PEX19-independent pathway. (A) The first 50 aa of PEX3 are sufficient for peroxisomal targeting. Wild-type human fibroblasts were transfected with a plasmid designed to express the PEX3 mPTS (PEX3aa1-50/6xmet3xmyc) and cells were processed for immunofluorescence using antibodies to the c-myc epitope (left) or PMP70 (right). Bar, 15 μM. (B) The mPTS of PEX3 does not coprecipitate with PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either 3xHA-PEX19 (+3xHA-PEX19) or PEX19 (+PEX19). Equal total protein from a membrane-free lysate of each cell sample was subjected to immunoprecipitation with antibodies to the HA epitope. IPs (top) and whole cell lysates (bottom) were processed for immunoblot using anti-myc antibodies. (C) The subcellular distribution of the PEX3 mPTS is not affected by PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either PEX19 (top) or 3xNLS-PEX19 (bottom). Cells were processed for indirect immunofluorescence using anti-myc (left) or anti-PEX19 (right) antibodies. Bar, 15 μM. (D) Inhibition of PEX19 does not affect peroxisomal targeting of the PEX3 mPTS. Wild-type human fibroblasts were subjected to electroporation with either TRIP8b siRNA (top) or PEX19 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and the PEX3 mPTS. Cells were processed for immunofluorescence using antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells importing HA-PTE1 were scored as to whether the PEX3 mPTS was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 311; PEX19 siRNA n = 294. Bar, 15 μM.

Mentions: The hypothesis that PEX19 is a PMP chaperone and import receptor contrasts with previous observations that PEX19 does not bind to the mPTS of PEX3, another integral PMP (Kammerer et al., 1998; Soukupova et al., 1999; Snyder et al., 2000; Fransen et al., 2001). Therefore, we tested whether PEX19 functioned as a chaperone and import receptor for PEX3 (Fig. 6). Using the same assays that showed strong interaction between PEX19 and other mPTSs, we found that the PEX3 mPTS (PEX3aa1-50/6xmet3xmyc) is neither bound by PEX19 (Fig. 6 B) nor is its subcellular distribution affected by PEX19 or 3xNLSPEX19 (Fig. 6 C). Rather, the PEX3 mPTS was targeted to mitochondria in cells that lack peroxisomes, as reported previously for PEX3 and the PEX3 mPTS (Soukupova et al., 1999; Sacksteder et al., 2000).


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)

The mPTS of PEX3 functions via a PEX19-independent pathway. (A) The first 50 aa of PEX3 are sufficient for peroxisomal targeting. Wild-type human fibroblasts were transfected with a plasmid designed to express the PEX3 mPTS (PEX3aa1-50/6xmet3xmyc) and cells were processed for immunofluorescence using antibodies to the c-myc epitope (left) or PMP70 (right). Bar, 15 μM. (B) The mPTS of PEX3 does not coprecipitate with PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either 3xHA-PEX19 (+3xHA-PEX19) or PEX19 (+PEX19). Equal total protein from a membrane-free lysate of each cell sample was subjected to immunoprecipitation with antibodies to the HA epitope. IPs (top) and whole cell lysates (bottom) were processed for immunoblot using anti-myc antibodies. (C) The subcellular distribution of the PEX3 mPTS is not affected by PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either PEX19 (top) or 3xNLS-PEX19 (bottom). Cells were processed for indirect immunofluorescence using anti-myc (left) or anti-PEX19 (right) antibodies. Bar, 15 μM. (D) Inhibition of PEX19 does not affect peroxisomal targeting of the PEX3 mPTS. Wild-type human fibroblasts were subjected to electroporation with either TRIP8b siRNA (top) or PEX19 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and the PEX3 mPTS. Cells were processed for immunofluorescence using antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells importing HA-PTE1 were scored as to whether the PEX3 mPTS was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 311; PEX19 siRNA n = 294. Bar, 15 μM.
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fig6: The mPTS of PEX3 functions via a PEX19-independent pathway. (A) The first 50 aa of PEX3 are sufficient for peroxisomal targeting. Wild-type human fibroblasts were transfected with a plasmid designed to express the PEX3 mPTS (PEX3aa1-50/6xmet3xmyc) and cells were processed for immunofluorescence using antibodies to the c-myc epitope (left) or PMP70 (right). Bar, 15 μM. (B) The mPTS of PEX3 does not coprecipitate with PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either 3xHA-PEX19 (+3xHA-PEX19) or PEX19 (+PEX19). Equal total protein from a membrane-free lysate of each cell sample was subjected to immunoprecipitation with antibodies to the HA epitope. IPs (top) and whole cell lysates (bottom) were processed for immunoblot using anti-myc antibodies. (C) The subcellular distribution of the PEX3 mPTS is not affected by PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either PEX19 (top) or 3xNLS-PEX19 (bottom). Cells were processed for indirect immunofluorescence using anti-myc (left) or anti-PEX19 (right) antibodies. Bar, 15 μM. (D) Inhibition of PEX19 does not affect peroxisomal targeting of the PEX3 mPTS. Wild-type human fibroblasts were subjected to electroporation with either TRIP8b siRNA (top) or PEX19 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and the PEX3 mPTS. Cells were processed for immunofluorescence using antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells importing HA-PTE1 were scored as to whether the PEX3 mPTS was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 311; PEX19 siRNA n = 294. Bar, 15 μM.
Mentions: The hypothesis that PEX19 is a PMP chaperone and import receptor contrasts with previous observations that PEX19 does not bind to the mPTS of PEX3, another integral PMP (Kammerer et al., 1998; Soukupova et al., 1999; Snyder et al., 2000; Fransen et al., 2001). Therefore, we tested whether PEX19 functioned as a chaperone and import receptor for PEX3 (Fig. 6). Using the same assays that showed strong interaction between PEX19 and other mPTSs, we found that the PEX3 mPTS (PEX3aa1-50/6xmet3xmyc) is neither bound by PEX19 (Fig. 6 B) nor is its subcellular distribution affected by PEX19 or 3xNLSPEX19 (Fig. 6 C). Rather, the PEX3 mPTS was targeted to mitochondria in cells that lack peroxisomes, as reported previously for PEX3 and the PEX3 mPTS (Soukupova et al., 1999; Sacksteder et al., 2000).

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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