Limits...
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.

Show MeSH
PEX19 binds the transmembrane region of the PMP34 mPTS. PEX3-deficient human fibroblasts expressing 3xNLS-PEX19 and myc-tagged mutant forms of the COOH-terminal PMP34 mPTS were processed for indirect immunofluorescence using antibodies to the myc epitope and PEX19. (A, left) Diagram showing amino acid range of each truncation mutant studied. Darkened regions indicated relative position of the putative transmembrane domain. Light vertical bars indicate relative positions of the engineered point mutations. All proteins fused to three tandem c-myc epitopes at the COOH terminus. (Right) Bar graph of relative binding efficiency of each mutant. Relative binding efficiency is the proportion of cells expressing both 3xNLS-PEX19 and PMP34 mPTS mutant in which the PMP34 mPTS mutant is seen in the nucleus. n = 100 cells for each sample. Values are normalized. (B) Amino acids 270-307 of PMP34. Putative transmembrane domain is boxed, sites of engineered point mutations are in bold and underlined. (C) Immunofluorescence images of cells expressing selected PMP34 mPTS mutants from the experiment in A. (Left) Anti-myc staining; (right) anti-PEX19 staining. Bar, 15 μM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171958&req=5

fig3: PEX19 binds the transmembrane region of the PMP34 mPTS. PEX3-deficient human fibroblasts expressing 3xNLS-PEX19 and myc-tagged mutant forms of the COOH-terminal PMP34 mPTS were processed for indirect immunofluorescence using antibodies to the myc epitope and PEX19. (A, left) Diagram showing amino acid range of each truncation mutant studied. Darkened regions indicated relative position of the putative transmembrane domain. Light vertical bars indicate relative positions of the engineered point mutations. All proteins fused to three tandem c-myc epitopes at the COOH terminus. (Right) Bar graph of relative binding efficiency of each mutant. Relative binding efficiency is the proportion of cells expressing both 3xNLS-PEX19 and PMP34 mPTS mutant in which the PMP34 mPTS mutant is seen in the nucleus. n = 100 cells for each sample. Values are normalized. (B) Amino acids 270-307 of PMP34. Putative transmembrane domain is boxed, sites of engineered point mutations are in bold and underlined. (C) Immunofluorescence images of cells expressing selected PMP34 mPTS mutants from the experiment in A. (Left) Anti-myc staining; (right) anti-PEX19 staining. Bar, 15 μM.

Mentions: The hypothesis that PMP import requires a bifunctional PMP chaperone/import receptor is rooted in the assumption that the hydrophobic transmembrane domains of PMPs must be masked as PMPs move through the cytoplasm to the peroxisome membrane. If this is true, and if PEX19 is the PMP chaperone/import receptor of this hypothesis, then PEX19 should bind to subregions of mPTSs that contain transmembrane domains. We tested this prediction using the COOH-terminal mPTS of PMP34, which contains a single transmembrane domain. We generated a series of mutations in this mPTS and examined their effects on PEX19 interaction in vivo. Truncation mutants that retained the transmembrane domain retained their interaction with PEX19, whereas those that lacked part of the transmembrane domain were no longer efficiently bound by PEX19 (Fig. 3). The smallest fragment that retained full interaction with PEX19, PMP34aa270-307, contains a long hydrophobic stretch that is interrupted by a pair of charged residues (E289 and K290). Replacement of these charged residues with leucines inhibited the fragment's interaction with PEX19, as did replacement of three hydrophobic residues with hydrophilic amino acids (LMF283-285KKK). Replacing the flanking basic residues at the COOH-terminal side of the putative transmembrane domain with acidic residues (KR302-303EE) had no substantive effect on mPTS–PEX19 interaction. These results indicate that PEX19 interacts with the transmembrane domain of the PMP34 mPTS, and that the distribution of hydrophobic and charged residues within the transmembrane domain are important for mPTS–PEX19 interaction.


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 the transmembrane region of the PMP34 mPTS. PEX3-deficient human fibroblasts expressing 3xNLS-PEX19 and myc-tagged mutant forms of the COOH-terminal PMP34 mPTS were processed for indirect immunofluorescence using antibodies to the myc epitope and PEX19. (A, left) Diagram showing amino acid range of each truncation mutant studied. Darkened regions indicated relative position of the putative transmembrane domain. Light vertical bars indicate relative positions of the engineered point mutations. All proteins fused to three tandem c-myc epitopes at the COOH terminus. (Right) Bar graph of relative binding efficiency of each mutant. Relative binding efficiency is the proportion of cells expressing both 3xNLS-PEX19 and PMP34 mPTS mutant in which the PMP34 mPTS mutant is seen in the nucleus. n = 100 cells for each sample. Values are normalized. (B) Amino acids 270-307 of PMP34. Putative transmembrane domain is boxed, sites of engineered point mutations are in bold and underlined. (C) Immunofluorescence images of cells expressing selected PMP34 mPTS mutants from the experiment in A. (Left) Anti-myc staining; (right) anti-PEX19 staining. Bar, 15 μM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171958&req=5

fig3: PEX19 binds the transmembrane region of the PMP34 mPTS. PEX3-deficient human fibroblasts expressing 3xNLS-PEX19 and myc-tagged mutant forms of the COOH-terminal PMP34 mPTS were processed for indirect immunofluorescence using antibodies to the myc epitope and PEX19. (A, left) Diagram showing amino acid range of each truncation mutant studied. Darkened regions indicated relative position of the putative transmembrane domain. Light vertical bars indicate relative positions of the engineered point mutations. All proteins fused to three tandem c-myc epitopes at the COOH terminus. (Right) Bar graph of relative binding efficiency of each mutant. Relative binding efficiency is the proportion of cells expressing both 3xNLS-PEX19 and PMP34 mPTS mutant in which the PMP34 mPTS mutant is seen in the nucleus. n = 100 cells for each sample. Values are normalized. (B) Amino acids 270-307 of PMP34. Putative transmembrane domain is boxed, sites of engineered point mutations are in bold and underlined. (C) Immunofluorescence images of cells expressing selected PMP34 mPTS mutants from the experiment in A. (Left) Anti-myc staining; (right) anti-PEX19 staining. Bar, 15 μM.
Mentions: The hypothesis that PMP import requires a bifunctional PMP chaperone/import receptor is rooted in the assumption that the hydrophobic transmembrane domains of PMPs must be masked as PMPs move through the cytoplasm to the peroxisome membrane. If this is true, and if PEX19 is the PMP chaperone/import receptor of this hypothesis, then PEX19 should bind to subregions of mPTSs that contain transmembrane domains. We tested this prediction using the COOH-terminal mPTS of PMP34, which contains a single transmembrane domain. We generated a series of mutations in this mPTS and examined their effects on PEX19 interaction in vivo. Truncation mutants that retained the transmembrane domain retained their interaction with PEX19, whereas those that lacked part of the transmembrane domain were no longer efficiently bound by PEX19 (Fig. 3). The smallest fragment that retained full interaction with PEX19, PMP34aa270-307, contains a long hydrophobic stretch that is interrupted by a pair of charged residues (E289 and K290). Replacement of these charged residues with leucines inhibited the fragment's interaction with PEX19, as did replacement of three hydrophobic residues with hydrophilic amino acids (LMF283-285KKK). Replacing the flanking basic residues at the COOH-terminal side of the putative transmembrane domain with acidic residues (KR302-303EE) had no substantive effect on mPTS–PEX19 interaction. These results indicate that PEX19 interacts with the transmembrane domain of the PMP34 mPTS, and that the distribution of hydrophobic and charged residues within the transmembrane domain are important for mPTS–PEX19 interaction.

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.

Show MeSH