The importance of peroxisomes is highlighted by severe inherited human disorders linked to impaired peroxisomal biogenesis. Besides the simple architecture of these ubiquitous and dynamic organelles, ...their biogenesis is surprisingly complex and involves specialized proteins, termed peroxins, which mediate targeting and insertion of peroxisomal membrane proteins (PMPs) into the peroxisomal bilayer, and the import of soluble proteins into the protein-dense matrix of the organelle. The long-standing paradigm that all peroxisomal proteins are imported directly into preexisting peroxisomes has been challenged by the detection of PMPs inside the endoplasmic reticulum (ER). New models propose that the ER originates peroxisomal biogenesis by mediating PMP trafficking to the peroxisomes via budding vesicles. However, the relative contribution of this ER-derived pathway to the total peroxisome population in vivo, and the detailed mechanisms of ER entry and exit of PMPs are controversially discussed. This review aims to summarize present knowledge about how PMPs are targeted to the ER, instead of being inserted directly into preexisting peroxisomes. Moreover, molecular mechanisms that facilitate bilayer insertion of PMPs among different species are discussed. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann.
•Peroxisomal membrane proteins (PMPs) may insert directly into preexisting peroxisomes.•PMPs may transit through the endoplasmic reticulum on their way to peroxisomes.•Peroxisomes are formed de novo and/or via growth and division.
Protein homeostasis results in a steady supply of peptides, which are further degraded to fuel protein synthesis or metabolic needs of the cell. In higher vertebrates, a small fraction of the ...resulting peptidome, however, is translocated into the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Antigenic peptides are guided to major histocompatibility complex class I (MHC I) molecules and are finally displayed on the cell surface, where they mount an adaptive immune response against viral infected or malignantly transformed cells. Here, we review the structural organization and the molecular mechanism of this specialized antigen translocon. We discuss how the ATP-binding cassette (ABC) transporter TAP communicates and cooperates within the multi-component peptide loading machinery, mediating the proper assembly and editing of kinetically stable peptide/MHC I complexes. In light of its important role within the MHC I antigen processing pathway, TAP is a prime target for viral immune evasion strategies, and we summarize how this antigen translocation machinery is sabotaged by viral factors. Finally, we compare TAP with other ABC systems that facilitate peptide translocation.
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•The heterodimeric transport complex TAP translocates peptides into the endoplasmic reticulum.•TAP cooperates within a multi-component MHC I peptide loading machinery.•The ABC transporter TAP is specifically inhibited by viral immune evasins.•ABC translocation machineries mediate intracellular peptide compartmentalization.
Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic ...compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
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•The inhibited Sec translocon adopts a conformation optimal for substrate engagement•The inhibitor mycolactone wedges open the lateral gate of Sec61α•Mycolactone blocks the path taken by the signal peptide during engagement•Resistance mutations are likely to operate by modulating translocon dynamics
Gérard et al. determine the structure of the mammalian Sec translocon in an inhibited state. Mycolactone holds the translocon in a conformation optimal for substrate engagement, wedging open the cytosolic side of the lateral gate, while also blocking the path taken by the signal peptide during engagement.
Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have evolved sophisticated mechanisms to suppress the host immune system. For instance, flavivirus infections were found to sabotage ...peroxisomes, organelles with an important role in innate immunity. The current model suggests that the capsid (C) proteins of DENV and ZIKV downregulate peroxisomes, ultimately resulting in reduced production of interferons by interacting with the host protein PEX19, a crucial chaperone in peroxisomal biogenesis. Here, we aimed to explore the importance of peroxisomes and the role of C interaction with PEX19 in the flavivirus life cycle. By infecting cells lacking peroxisomes we show that this organelle is required for optimal DENV replication. Moreover, we demonstrate that DENV and ZIKV C bind PEX19 through a conserved PEX19-binding motif, which is also commonly found in cellular peroxisomal membrane proteins (PMPs). However, in contrast to PMPs, this interaction does not result in the targeting of C to peroxisomes. Furthermore, we show that the presence of C results in peroxisome loss due to impaired peroxisomal biogenesis, which appears to occur by a PEX19-independent mechanism. Hence, these findings challenge the current model of how flavivirus C might downregulate peroxisomal abundance and suggest a yet unknown role of peroxisomes in flavivirus biology.
The long‐standing paradigm that all peroxisomal proteins are imported post‐translationally into pre‐existing peroxisomes has been challenged by the detection of peroxisomal membrane proteins (PMPs) ...inside the endoplasmic reticulum (ER). In mammals, the mechanisms of ER entry and exit of PMPs are completely unknown. We show that the human PMP PEX3 inserts co‐translationally into the mammalian ER via the Sec61 translocon. Photocrosslinking and fluorescence spectroscopy studies demonstrate that the N‐terminal transmembrane segment (TMS) of ribosome‐bound PEX3 is recognized by the signal recognition particle (SRP). Binding to SRP is a prerequisite for targeting of the PEX3‐containing ribosome•nascent chain complex (RNC) to the translocon, where an ordered multistep pathway integrates the nascent chain into the membrane adjacent to translocon proteins Sec61α and TRAM. This insertion of PEX3 into the ER is physiologically relevant because PEX3 then exits the ER via budding vesicles in an ATP‐dependent process. This study identifies early steps in human peroxisomal biogenesis by demonstrating sequential stages of PMP passage through the mammalian ER.
Some peroxisomal membrane proteins (PMPs) are targeted first to the endoplasmic reticulum (ER) prior to being transported to peroxisomes. The mechanisms that mediate ER‐passage of PMPs in mammalians are unknown. We demonstrate that the human PMP PEX3 inserts co‐translationally into the ER by an SRP/Sec61‐translocon‐dependent multistep pathway. Subsequently, PEX3 exits the ER via budding vesicles in an energy‐consuming reaction. Hence, we demonstrate how the ER is seeded with PMPs, and highlight the significance of ER‐to‐peroxisome vesicle trafficking in human organelle biogenesis.
Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have ...evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors.
Background: Herpesviruses have evolved sophisticated strategies to escape immune surveillance.
Results: EBV BNLF2a acts as tail-anchored protein and is posttranslationally inserted into the ER membrane, where it arrests core TAP in a transport-incompetent conformation.
Conclusion: BNLF2a exploits the host tail-anchored protein insertion machinery.
Significance: This inhibition mechanism is distinct and mutually exclusive of other viral TAP inhibitors.
After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ...ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 °C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 °C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 °C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.
Zellweger cerebro-hepato-renal syndrome is a severe congenital disorder associated with defective peroxisomal biogenesis. At least 23
PEX genes have been reported to be essential for peroxisome ...biogenesis in various species, indicating the complexity of peroxisomal assembly. Cells from patients with peroxisomal biogenesis disorders have previously been shown to segregate into ≥12 complementation groups. Two patients assigned to complementation group G who had not been linked previously to a specific gene defect were confirmed as displaying a cellular phenotype characterized by a lack of even residual peroxisomal membrane structures. Here we demonstrate that this complementation group is associated with mutations in the
PEX3 gene, encoding an integral peroxisomal membrane protein. Homozygous
PEX3 mutations, each leading to C-terminal truncation of PEX3, were identified in the two patients, who both suffered from a severe Zellweger syndrome phenotype. One of the mutations involved a single-nucleotide insertion in exon 7, whereas the other was a single-nucleotide substitution eight nucleotides from the normal splice site in the 3′ acceptor site of intron 10. Expression of wild-type PEX3 in the mutant cell lines restored peroxisomal biogenesis, whereas transfection of mutated
PEX3 cDNA did not. This confirmed that the causative gene had been identified. The observation of peroxisomal formation in the absence of morphologically recognizable peroxisomal membranes challenges the theory that peroxisomes arise exclusively by growth and division from preexisting peroxisomes and establishes
PEX3 as a key factor in early human peroxisome synthesis.
Strikingly variable clinical phenotypes can be found in X-linked adrenoleukodystrophy (X-ALD) even with the same
ABCD1 mutation.
ABCD2 is the closest homolog to
ABCD1. Since
ABCD2 overexpression ...complements the loss of
ABCD1 in vivo and
in vitro, we have investigated the possible role of the
ABCD2 gene locus as determinant of X-ALD phenotypes. Sequence and segregation analysis of the
ABCD2 gene, in a large X-ALD family with different phenotypes disclosed that the identical
ABCD2 alleles were inherited in brothers affected by mild (noncerebral) versus severe (childhood cerebral) X-ALD phenotypes. Moreover, two independent association studies of
ABCD2 polymorphisms and clinical phenotypes showed an even allele distribution in different X-ALD phenotypes and controls. Based on these findings
ABCD2 can be excluded as a major modifier locus for clinical diversity in X-ALD. These findings are of particular importance for the attempt of pharmacological induction of
ABCD2 as a possible therapeutic approach in X-ALD.
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