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Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p.

ABSTRACT: Peroxisomes require the translocation of folded and functional target proteins of various sizes across the peroxisomal membrane. We have investigated the structure and function of the principal import receptor Pex5p, which recognizes targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal structures of the receptor in the presence and absence of a peroxisomal target, sterol carrier protein 2, reveal major structural changes from an open, snail-like conformation into a closed, circular conformation. These changes are caused by a long loop C terminal to the 7-fold tetratricopeptide repeat segments. Mutations in residues of this loop lead to defects in peroxisomal import in human fibroblasts. The structure of the receptor/cargo complex demonstrates that the primary receptor-binding site of the cargo is structurally and topologically autonomous, enabling the cargo to retain its native structure and function.

SUBMITTER: Stanley WA

PROVIDER: S-EPMC5030714 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p.

Stanley Will A WA Filipp Fabian V FV Kursula Petri P Schüller Nicole N Erdmann Ralf R Schliebs Wolfgang W Sattler Michael M Wilmanns Matthias M

Molecular cell 20061201 5

Peroxisomes require the translocation of folded and functional target proteins of various sizes across the peroxisomal membrane. We have investigated the structure and function of the principal import receptor Pex5p, which recognizes targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal structures of the receptor in the presence and absence of a peroxisomal target, sterol carrier protein 2, reveal major structural changes from an open, snail-like conformation into a closed, c ...[more]

PMID: 17157249

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Project description:Unlike most organellar proteins, some peroxisomal proteins are often found in significant amounts in the cytosol. Such apparent import inefficiency is very marked in guinea pig (Cavia porcellus) hepatocytes in which the cytosolic levels of two peroxisomal proteins, catalase and alanine:glyoxylate aminotransferase (AGT), are much higher than those found in human (Homo sapiens) hepatocytes, for example. In an attempt to provide an explanation for this phenomenon, we have cloned the guinea pig CpPEX5 gene, which encodes the peroxisomal targeting sequence type 1 (PTS1) import receptor Pex5p, and functionally compared it with its human homologue, HsPex5p. Our results showed the following: (1) CpPEX5, like its human homologue, encodes two splice variants differing by the presence or absence of an internal region of 37 amino acids; (2) both variants were expressed in all guinea pig tissues studied; (3) both variants were equally able to complement peroxisomal import of PTS1 proteins in microinjected Deltapex5 human fibroblasts; (4) CpPex5p was as efficient as HsPex5p in mediating the peroxisomal import of proteins possessing the consensus PTS1, Ser-Lys-Leu, but much less efficient in mediating the import of proteins possessing non-consensus PTS1s (i.e. Lys-Lys-Leu of human AGT and Ala-Asn-Leu of human catalase); (5) reporter proteins with the consensus PTS1, Ser-Lys-Leu, inhibited the peroxisomal import of endogenous catalase, whereas AGT with the non-consensus Lys-Lys-Leu did not; (6) high concentrations of HsPex5p, but not CpPex5p, markedly inhibited the import of AGT, but not catalase or proteins ending in Ser-Lys-Leu; and (7) in the yeast two-hybrid system, AGT-Ser-Lys-Leu interacted with the tetratricopeptide repeat domain of HsPex5p, but AGT-Lys-Lys-Leu did not. In addition, AGT-Ser-Lys-Leu was targeted to peroxisomes in Saccharomyces cerevisiae, whereas AGT-Lys-Lys-Leu was not. These data suggest that the inefficient peroxisomal import of AGT and catalase in guinea pig cells is due to the inefficiency with which CpPex5p mediates the peroxisomal import of proteins containing non-consensus PTS1s. They also suggest that the non-consensus PTS1 of human AGT might interact with HsPex5p very differently compared with the consensus PTS1, Ser-Lys-Leu.

Project description:Peroxisome biogenesis diseases (PBDs) are characterized by global defects in peroxisomal function and can result in severe brain, liver, kidney, and bone malfunctions. PBDs are due to mutations in peroxisome biogenesis factors (PEX genes) that are responsible for peroxisome assembly and function. Increasing evidence suggests that peroxisome import functions decline during aging. However, the transcriptome profiling of peroxisome import defects and how they affect disease development are still lacking. PEX5 encodes the cytoplasmic receptors for peroxisome-targeting signal types 1. We generate knock-in human HEK293 cells mutant using CRISPR to transiently express PEX5 cysteine 11 to alanine mutant (PEX5C11A), which blocks PEX5 recycling and exerts dominant negative effect on PEX5 mediated peroxisome import. To identify conserved responses, we perform transcriptomic analysis on Drosophila oenocyte-specific Pex1, Pex12 and Pex5 knockdowns and on human cells with impaired peroxisome import (through expressing PEX5C11A and applying PEX5 siRNA respectively). PEX5C11A induction triggers vast transcriptomic changes, including decreased oxidative phosphorylation, increased MAPK signaling and HIPPO signaling. PEX5 siRNA specifically decreases spliceosome activity and increases cholesterol metabolism. Using gene set enrichment analysis (GSEA), we identify protein processing in endoplasmic reticulum pathway, specifically ER-associated protein degradation (ERAD) pathway is induced in all PEX knockdowns in Drosophila. Peroxisome dysfunction elevates eIF2α phosphorylation in both Drosophila and human cell culture independent of XBP1 activation, suggesting increased integrative stress response (ISR). Moreover, peroxisome stress decreases ribosome biogenesis genes and impairs ribosome biogenesis in flies and human cells. Specifically, peroxisome stress impairs the 5'-ETS cleavage activity during the ribosome biogenesis and dampens 40S small ribosomal export in both flies and human. Our results suggest that reduced ribosome biogenesis and elevated ISR could be conserved cellular response to peroxisome import stress.

Dataset Information

Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p.

Publications

Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p.

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