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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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Pex19 is a PMP chaperone. (A) PEX19 expression results in increased abundance of PMPs in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34myc, PEX11βmyc, PMP24/3xmyc, VSV-G, mycPTE1, and PEX19 (+PEX19). PEX19-deficient human fibroblasts were transfected with plasmids designed to express the same test proteins and vector alone (−PEX19). Equal total protein from a membrane-free lysate of each cell sample was analyzed by immunoblot using antibodies to the c-myc epitope or to VSV-G as appropriate. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 and either PMP34myc, PEX11βmyc, or PMP24/3xmyc were processed for indirect immunofluorescence using antibodies to the c-myc epitope (left) or PEX19 (right). Bar, 15 μM. (C) PEX19 stabilizes PMP34/13xmyc in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34/13xmyc and PEX19 (+PEX19); PEX19-deficient human fibroblasts were transfected with a plasmid designed to express PMP34/13xmyc and empty vector (−PEX19). Cells were pulsed for 15 min with [35S]methionine and chased with excess cold methionine for the times indicated. A membrane-free cell lysate of each sample was subjected to immunoprecipitation with anti-myc antibodies. Equal fractions of each IP were analyzed by autoradiography (35S) and immunoblot using anti-myc antibodies (anti-myc). (D) Relative 35S-signal intensities for each time point in B versus time with best fit exponential curves. (□, solid line), +PEX19; (♦, dashed line), −PEX19; t1/2 (+PEX19) = 300 min; t1/2 (−PEX19) = 15 min. (E) PEX19 binds multiple PMPs in the cytosol. PEX3-deficient human fibroblasts transfected with plasmids designed to express PMP34/13xmyc, PEX11βmyc, PMP24/3xmyc, 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 anti-HA antibodies. IPs were analyzed by immunoblot using anti-myc antibodies. (F) The majority of cytosolic PMP is bound to PEX19. Equal amounts of lysates of 3xHA-PEX19–expressing cells from E sampled before (Pre-IP) and after (Post-IP) immunoprecipitation were analyzed by immunoblot using anti-myc antibodies. (G) PEX19 interacts with newly synthesized PMPs. PEX19-deficient human fibroblasts stably expressing 3xHA-PEX19 were transfected with a plasmid designed to express PMP34/13xmyc. Cells were pulsed with [35S]methionine for 10 min, chased with excess methionine for the indicated times, and split into two fractions. Membrane-free lysates of cells in the first fraction were subjected to immunoprecipitation with anti-HA antibodies. The samples were solubilized in SDS and subjected to a second immunoprecipitation with anti-myc antibodies. Immunoprecipitations were analyzed by autoradiography (PEX19-associated, 35S) and immunoblot using anti-myc antibodies (PEX19-associated, anti-myc). Cells from the second fraction were solubilized in 1% Triton X-100 and subjected to immunoprecipitation using anti-myc antibodies; and PMP34/13xmyc was detected by autoradiography (whole-cell IP, 35S). For PEX19-PMP34/13xmyc association, t1/2 = 15 min. For the lifetime of PMP34/13xmyc in the whole cell, t1/2 = 300 min.
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fig1: Pex19 is a PMP chaperone. (A) PEX19 expression results in increased abundance of PMPs in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34myc, PEX11βmyc, PMP24/3xmyc, VSV-G, mycPTE1, and PEX19 (+PEX19). PEX19-deficient human fibroblasts were transfected with plasmids designed to express the same test proteins and vector alone (−PEX19). Equal total protein from a membrane-free lysate of each cell sample was analyzed by immunoblot using antibodies to the c-myc epitope or to VSV-G as appropriate. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 and either PMP34myc, PEX11βmyc, or PMP24/3xmyc were processed for indirect immunofluorescence using antibodies to the c-myc epitope (left) or PEX19 (right). Bar, 15 μM. (C) PEX19 stabilizes PMP34/13xmyc in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34/13xmyc and PEX19 (+PEX19); PEX19-deficient human fibroblasts were transfected with a plasmid designed to express PMP34/13xmyc and empty vector (−PEX19). Cells were pulsed for 15 min with [35S]methionine and chased with excess cold methionine for the times indicated. A membrane-free cell lysate of each sample was subjected to immunoprecipitation with anti-myc antibodies. Equal fractions of each IP were analyzed by autoradiography (35S) and immunoblot using anti-myc antibodies (anti-myc). (D) Relative 35S-signal intensities for each time point in B versus time with best fit exponential curves. (□, solid line), +PEX19; (♦, dashed line), −PEX19; t1/2 (+PEX19) = 300 min; t1/2 (−PEX19) = 15 min. (E) PEX19 binds multiple PMPs in the cytosol. PEX3-deficient human fibroblasts transfected with plasmids designed to express PMP34/13xmyc, PEX11βmyc, PMP24/3xmyc, 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 anti-HA antibodies. IPs were analyzed by immunoblot using anti-myc antibodies. (F) The majority of cytosolic PMP is bound to PEX19. Equal amounts of lysates of 3xHA-PEX19–expressing cells from E sampled before (Pre-IP) and after (Post-IP) immunoprecipitation were analyzed by immunoblot using anti-myc antibodies. (G) PEX19 interacts with newly synthesized PMPs. PEX19-deficient human fibroblasts stably expressing 3xHA-PEX19 were transfected with a plasmid designed to express PMP34/13xmyc. Cells were pulsed with [35S]methionine for 10 min, chased with excess methionine for the indicated times, and split into two fractions. Membrane-free lysates of cells in the first fraction were subjected to immunoprecipitation with anti-HA antibodies. The samples were solubilized in SDS and subjected to a second immunoprecipitation with anti-myc antibodies. Immunoprecipitations were analyzed by autoradiography (PEX19-associated, 35S) and immunoblot using anti-myc antibodies (PEX19-associated, anti-myc). Cells from the second fraction were solubilized in 1% Triton X-100 and subjected to immunoprecipitation using anti-myc antibodies; and PMP34/13xmyc was detected by autoradiography (whole-cell IP, 35S). For PEX19-PMP34/13xmyc association, t1/2 = 15 min. For the lifetime of PMP34/13xmyc in the whole cell, t1/2 = 300 min.

Mentions: Immunoblot analysis revealed that integral PMPs were much more abundant in peroxisome-deficient cells that express PEX19 than in cells that do not express PEX19 (Fig. 1 A). These results reflect the status of soluble, cytoplasmic PMPs in detergent-free, membrane-free lysates. This was confirmed by immunofluorescence studies which revealed that all three PMPs shared the same cytoplasmic accumulation and nuclear exclusion as PEX19 (Fig. 1 B). In contrast, these PMPs could only rarely be detected in PEX19-deficient cells, and what little could be detected was mislocalized to the mitochondrion (unpublished data).


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)

Pex19 is a PMP chaperone. (A) PEX19 expression results in increased abundance of PMPs in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34myc, PEX11βmyc, PMP24/3xmyc, VSV-G, mycPTE1, and PEX19 (+PEX19). PEX19-deficient human fibroblasts were transfected with plasmids designed to express the same test proteins and vector alone (−PEX19). Equal total protein from a membrane-free lysate of each cell sample was analyzed by immunoblot using antibodies to the c-myc epitope or to VSV-G as appropriate. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 and either PMP34myc, PEX11βmyc, or PMP24/3xmyc were processed for indirect immunofluorescence using antibodies to the c-myc epitope (left) or PEX19 (right). Bar, 15 μM. (C) PEX19 stabilizes PMP34/13xmyc in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34/13xmyc and PEX19 (+PEX19); PEX19-deficient human fibroblasts were transfected with a plasmid designed to express PMP34/13xmyc and empty vector (−PEX19). Cells were pulsed for 15 min with [35S]methionine and chased with excess cold methionine for the times indicated. A membrane-free cell lysate of each sample was subjected to immunoprecipitation with anti-myc antibodies. Equal fractions of each IP were analyzed by autoradiography (35S) and immunoblot using anti-myc antibodies (anti-myc). (D) Relative 35S-signal intensities for each time point in B versus time with best fit exponential curves. (□, solid line), +PEX19; (♦, dashed line), −PEX19; t1/2 (+PEX19) = 300 min; t1/2 (−PEX19) = 15 min. (E) PEX19 binds multiple PMPs in the cytosol. PEX3-deficient human fibroblasts transfected with plasmids designed to express PMP34/13xmyc, PEX11βmyc, PMP24/3xmyc, 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 anti-HA antibodies. IPs were analyzed by immunoblot using anti-myc antibodies. (F) The majority of cytosolic PMP is bound to PEX19. Equal amounts of lysates of 3xHA-PEX19–expressing cells from E sampled before (Pre-IP) and after (Post-IP) immunoprecipitation were analyzed by immunoblot using anti-myc antibodies. (G) PEX19 interacts with newly synthesized PMPs. PEX19-deficient human fibroblasts stably expressing 3xHA-PEX19 were transfected with a plasmid designed to express PMP34/13xmyc. Cells were pulsed with [35S]methionine for 10 min, chased with excess methionine for the indicated times, and split into two fractions. Membrane-free lysates of cells in the first fraction were subjected to immunoprecipitation with anti-HA antibodies. The samples were solubilized in SDS and subjected to a second immunoprecipitation with anti-myc antibodies. Immunoprecipitations were analyzed by autoradiography (PEX19-associated, 35S) and immunoblot using anti-myc antibodies (PEX19-associated, anti-myc). Cells from the second fraction were solubilized in 1% Triton X-100 and subjected to immunoprecipitation using anti-myc antibodies; and PMP34/13xmyc was detected by autoradiography (whole-cell IP, 35S). For PEX19-PMP34/13xmyc association, t1/2 = 15 min. For the lifetime of PMP34/13xmyc in the whole cell, t1/2 = 300 min.
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fig1: Pex19 is a PMP chaperone. (A) PEX19 expression results in increased abundance of PMPs in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34myc, PEX11βmyc, PMP24/3xmyc, VSV-G, mycPTE1, and PEX19 (+PEX19). PEX19-deficient human fibroblasts were transfected with plasmids designed to express the same test proteins and vector alone (−PEX19). Equal total protein from a membrane-free lysate of each cell sample was analyzed by immunoblot using antibodies to the c-myc epitope or to VSV-G as appropriate. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 and either PMP34myc, PEX11βmyc, or PMP24/3xmyc were processed for indirect immunofluorescence using antibodies to the c-myc epitope (left) or PEX19 (right). Bar, 15 μM. (C) PEX19 stabilizes PMP34/13xmyc in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34/13xmyc and PEX19 (+PEX19); PEX19-deficient human fibroblasts were transfected with a plasmid designed to express PMP34/13xmyc and empty vector (−PEX19). Cells were pulsed for 15 min with [35S]methionine and chased with excess cold methionine for the times indicated. A membrane-free cell lysate of each sample was subjected to immunoprecipitation with anti-myc antibodies. Equal fractions of each IP were analyzed by autoradiography (35S) and immunoblot using anti-myc antibodies (anti-myc). (D) Relative 35S-signal intensities for each time point in B versus time with best fit exponential curves. (□, solid line), +PEX19; (♦, dashed line), −PEX19; t1/2 (+PEX19) = 300 min; t1/2 (−PEX19) = 15 min. (E) PEX19 binds multiple PMPs in the cytosol. PEX3-deficient human fibroblasts transfected with plasmids designed to express PMP34/13xmyc, PEX11βmyc, PMP24/3xmyc, 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 anti-HA antibodies. IPs were analyzed by immunoblot using anti-myc antibodies. (F) The majority of cytosolic PMP is bound to PEX19. Equal amounts of lysates of 3xHA-PEX19–expressing cells from E sampled before (Pre-IP) and after (Post-IP) immunoprecipitation were analyzed by immunoblot using anti-myc antibodies. (G) PEX19 interacts with newly synthesized PMPs. PEX19-deficient human fibroblasts stably expressing 3xHA-PEX19 were transfected with a plasmid designed to express PMP34/13xmyc. Cells were pulsed with [35S]methionine for 10 min, chased with excess methionine for the indicated times, and split into two fractions. Membrane-free lysates of cells in the first fraction were subjected to immunoprecipitation with anti-HA antibodies. The samples were solubilized in SDS and subjected to a second immunoprecipitation with anti-myc antibodies. Immunoprecipitations were analyzed by autoradiography (PEX19-associated, 35S) and immunoblot using anti-myc antibodies (PEX19-associated, anti-myc). Cells from the second fraction were solubilized in 1% Triton X-100 and subjected to immunoprecipitation using anti-myc antibodies; and PMP34/13xmyc was detected by autoradiography (whole-cell IP, 35S). For PEX19-PMP34/13xmyc association, t1/2 = 15 min. For the lifetime of PMP34/13xmyc in the whole cell, t1/2 = 300 min.
Mentions: Immunoblot analysis revealed that integral PMPs were much more abundant in peroxisome-deficient cells that express PEX19 than in cells that do not express PEX19 (Fig. 1 A). These results reflect the status of soluble, cytoplasmic PMPs in detergent-free, membrane-free lysates. This was confirmed by immunofluorescence studies which revealed that all three PMPs shared the same cytoplasmic accumulation and nuclear exclusion as PEX19 (Fig. 1 B). In contrast, these PMPs could only rarely be detected in PEX19-deficient cells, and what little could be detected was mislocalized to the mitochondrion (unpublished data).

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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