Language：English   Publishing type：Research paper (scientific journal)  

Correct targeting of peroxisomal membrane proteins (PMPs) is essential for the formation and maintenance of functional peroxisomes. Activities of Pex19p to interact with PMPs on one hand and Pex3p on the other, including formation of ternary complexes between Pex19p, PMP and Pex3p, strongly support posttranslational translocation of PMPs via the Pex19p- and Pex3p-dependent direct pathway, termed the class I pathway. However, it remains elusive whether Pex19p-PMP complexes are indeed capable of being imported into peroxisomal membranes through the interaction between Pex19p and Pex3p. We resolve this issue by investigating the targeting process of several topologically distinct PMPs, including multimembrane spanning PMPs. We show here that Pex19p forms cytosolic complexes with PMPs and directly translocates them to peroxisomes. Using a semi-intact mammalian cell-based import assay system, we prove that PMPs in the cytosolic complexes are imported into peroxisomes via the interaction between cargo-loaded Pex19p and Pex3p. Furthermore, we demonstrate for the first time that peroxisomal targeting of ATAD1, an N-terminally signal-anchored protein that resides on both mitochondria and peroxisomes, is also achieved through the Pex19p- and Pex3p-dependent class I pathway. Together, our results suggest that translocation of PMPs via the class I pathway is a common event in mammalian cells.

DOI： 10.1111/tra.12376

Web of Science

Scopus

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Tail-anchored (TA) proteins are anchored into cellular membranes by a single transmembrane domain (TMD) close to the C terminus. Although the targeting of TA proteins to peroxisomes is dependent on PEX19, the mechanistic details of PEX19-dependent targeting and the signal that directs TA proteins to peroxisomes have remained elusive, particularly in mammals. The present study shows that PEX19 formed a complex with the peroxisomal TA protein PEX26 in the cytosol and translocated it directly to peroxisomes by interacting with the peroxisomal membrane protein PEX3. Unlike in yeast, the adenosine triphosphatase TRC40, which delivers TA proteins to the endoplasmic reticulum, was dispensable for the peroxisomal targeting of PEX26. Moreover, the basic amino acids within the luminal domain of PEX26 were essential for binding to PEX19 and thereby for peroxisomal targeting. Finally, our results suggest that a TMD that escapes capture by TRC40 and is followed by a highly basic luminal domain directs TA proteins to peroxisomes via the PEX19-dependent route. © 2013 Yagita et al.

DOI： 10.1083/jcb.201211077

Web of Science

Scopus

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication. © 2013, American Society for Microbiology.

DOI： 10.1128/JVI.02572-12

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS). Gene defects of peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic peroxins for ten complementation groups of PBDs, are required for matrix protein import; three, Pex3p, Pex16p and Pex19p, are shown to be essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three complementation groups 12, 9, and 14, respectively. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts and deletions. With respect to the function of these three peroxins in membrane biogenesis, two distinct pathways have been proposed for the import of peroxisomal membrane proteins in mammalian cells: a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex16p-dependent class II pathway. In class II pathway, Pex19p also forms a soluble complex with newly synthesized Pex3p as the chaperone for Pex3p in the cytosol and directly translocates it to peroxisomes. Pex16p functions as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. A model for the import of peroxisomal membrane proteins is suggested, providing new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p. Another model suggests that in Saccharomyces cerevisiae peroxisomes likely emerge from the endoplasmic reticulum. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease. © 2012 Elsevier B.V..

DOI： 10.1016/j.bbadis.2012.06.004

Web of Science

Scopus

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

We herein isolated plasmalogen-deficient Chinese hamster ovary (CHO) mutant, ZPEG251, with a phenotype of normal import of peroxisomal matrix and membrane proteins. In ZPEG251, plasmenylethanolamine (PlsEtn) was severely reduced. Complementation analysis by expression of genes responsible for the plasmalogen biogenesis suggested that alkyl-dihydroxyacetonephosphate synthase (ADAPS), catalyzing the second step of plasmalogen biogenesis, was deficient in ZPEG251. ADAPS mRNA was barely detectable as verified by Northern blot and reverse transcription-PCR analyses. Defect of ADAPS expression was also assessed by immunoblot. As a step toward delineating functional roles of PlsEtn, we investigated its subcellular localization. PlsEtn was localized to post-Golgi compartments and enriched in detergent-resistant membranes. Transport of PlsEtn to post-Golgi compartments was apparently affected by lowering cellular ATP, but not by inhibitors of microtubule assembly and vesicular transport. Partitioning of cholesterol and sphingomyelin, a typical feature of lipid rafts, was not impaired in plasmalogen-deficient cells, including peroxisome assembly-defective mutants, hence suggesting that PlsEtn was not essential for lipid-raft architecture in CHO cells. © 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamcr.2008.05.018

Web of Science

Scopus

PubMed

researchmap