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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 binds multiple PMP targeting signals. (A) Multiple mPTSs coprecipitate with PEX19. PEX3- deficient human fibroblasts were transfected with plasmids designed to express PMP34aa244-307/3xmyc, PEX11βaa181-259/3xmyc, PEX16aa221-336/3xmyc, PEX16aa59-219/3xmyc, PMP22aa1-94/3xmyc, PMP22aa95-195/3xmyc, PMP70aa1-61/13xmyc, PMP70aa61-160/13xmyc, PMP70aa1-124/3xmyc, 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. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using antibodies against PEX19. Bar, 15 μM. (C) PEX19 controls the subcellular distribution of multiple mPTSs. PEX3- deficient human fibroblasts transfected with plasmids designed to express the mPTSs from A and either PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using anti-myc antibodies. Bar, 15 μM.
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fig2: PEX19 binds multiple PMP targeting signals. (A) Multiple mPTSs coprecipitate with PEX19. PEX3- deficient human fibroblasts were transfected with plasmids designed to express PMP34aa244-307/3xmyc, PEX11βaa181-259/3xmyc, PEX16aa221-336/3xmyc, PEX16aa59-219/3xmyc, PMP22aa1-94/3xmyc, PMP22aa95-195/3xmyc, PMP70aa1-61/13xmyc, PMP70aa61-160/13xmyc, PMP70aa1-124/3xmyc, 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. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using antibodies against PEX19. Bar, 15 μM. (C) PEX19 controls the subcellular distribution of multiple mPTSs. PEX3- deficient human fibroblasts transfected with plasmids designed to express the mPTSs from A and either PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using anti-myc antibodies. Bar, 15 μM.

Mentions: Our hypothesis that PMP import requires a bifunctional PMP chaperone/import receptor led us to next ask whether PEX19 also functioned as a PMP import receptor. We first tested whether PEX19 binds to the mPTSs of multiple PMPs. We tested the c-terminal mPTS of PMP34 (Jones et al., 2001), an mPTS from PEX11β (Sacksteder et al., 2000), and both reported mPTSs of PMP22 (Brosius et al., 2002). We also tested two distinct mPTSs from PEX16, one reported previously (Fransen et al., 2001) and one (amino acids 221–336) identified in our laboratory (unpublished data). Finally, we examined three reported mPTSs of PMP70 (Sacksteder et al., 2000; Biermanns and Gartner, 2001). PEX3-deficient PBD400-TI cells, which lack peroxisomes, were cotransfected with plasmids designed to express the mPTS-containing proteins together with either PEX19 or 3xHA-PEX19. The next day the cells were lysed in hypotonic buffer and membranes were discarded after centrifugation. The resulting soluble protein lysates were subjected to immunoprecipitation with antibodies to the HA epitope tag, followed by immunoblot with antibodies specific for the mPTS-containing proteins. Each mPTS-containing protein was precipitated with 3xHA-PEX19, demonstrating the ability of PEX19 to bind these diverse mPTSs (Fig. 2 A).


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 binds multiple PMP targeting signals. (A) Multiple mPTSs coprecipitate with PEX19. PEX3- deficient human fibroblasts were transfected with plasmids designed to express PMP34aa244-307/3xmyc, PEX11βaa181-259/3xmyc, PEX16aa221-336/3xmyc, PEX16aa59-219/3xmyc, PMP22aa1-94/3xmyc, PMP22aa95-195/3xmyc, PMP70aa1-61/13xmyc, PMP70aa61-160/13xmyc, PMP70aa1-124/3xmyc, 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. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using antibodies against PEX19. Bar, 15 μM. (C) PEX19 controls the subcellular distribution of multiple mPTSs. PEX3- deficient human fibroblasts transfected with plasmids designed to express the mPTSs from A and either PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using anti-myc antibodies. Bar, 15 μM.
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fig2: PEX19 binds multiple PMP targeting signals. (A) Multiple mPTSs coprecipitate with PEX19. PEX3- deficient human fibroblasts were transfected with plasmids designed to express PMP34aa244-307/3xmyc, PEX11βaa181-259/3xmyc, PEX16aa221-336/3xmyc, PEX16aa59-219/3xmyc, PMP22aa1-94/3xmyc, PMP22aa95-195/3xmyc, PMP70aa1-61/13xmyc, PMP70aa61-160/13xmyc, PMP70aa1-124/3xmyc, 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. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using antibodies against PEX19. Bar, 15 μM. (C) PEX19 controls the subcellular distribution of multiple mPTSs. PEX3- deficient human fibroblasts transfected with plasmids designed to express the mPTSs from A and either PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using anti-myc antibodies. Bar, 15 μM.
Mentions: Our hypothesis that PMP import requires a bifunctional PMP chaperone/import receptor led us to next ask whether PEX19 also functioned as a PMP import receptor. We first tested whether PEX19 binds to the mPTSs of multiple PMPs. We tested the c-terminal mPTS of PMP34 (Jones et al., 2001), an mPTS from PEX11β (Sacksteder et al., 2000), and both reported mPTSs of PMP22 (Brosius et al., 2002). We also tested two distinct mPTSs from PEX16, one reported previously (Fransen et al., 2001) and one (amino acids 221–336) identified in our laboratory (unpublished data). Finally, we examined three reported mPTSs of PMP70 (Sacksteder et al., 2000; Biermanns and Gartner, 2001). PEX3-deficient PBD400-TI cells, which lack peroxisomes, were cotransfected with plasmids designed to express the mPTS-containing proteins together with either PEX19 or 3xHA-PEX19. The next day the cells were lysed in hypotonic buffer and membranes were discarded after centrifugation. The resulting soluble protein lysates were subjected to immunoprecipitation with antibodies to the HA epitope tag, followed by immunoblot with antibodies specific for the mPTS-containing proteins. Each mPTS-containing protein was precipitated with 3xHA-PEX19, demonstrating the ability of PEX19 to bind these diverse mPTSs (Fig. 2 A).

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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