scholarly journals Hydrophobic Regions Adjacent to Transmembrane Domains 1 and 5 Are Important for the Targeting of the 70-kDa Peroxisomal Membrane Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33831-33844 ◽  
Author(s):  
Yoshinori Kashiwayama ◽  
Kota Asahina ◽  
Masashi Morita ◽  
Tsuneo Imanaka
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Transmembrane Domains ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH2-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH2-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH2-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile307/Leu308) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.

scholarly journals The Peroxin Pex3p Initiates Membrane Assembly in Peroxisome Biogenesis

2000 ◽  
Vol 11 (6) ◽  
pp. 2085-2102 ◽  
Author(s):  
Kamran Ghaedi ◽  
Shigehiko Tamura ◽  
Kanji Okumoto ◽  
Yuji Matsuzono ◽  
Yukio Fujiki
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Membrane Vesicles ◽  
Integral Membrane Protein ◽  
Amino Acid Identity ◽  
Complementation Group ◽  
Ovary Cell ◽  
Peroxisomal Membrane

Rat cDNA encoding a 372-amino-acid peroxin was isolated, primarily by functional complementation screening, using a peroxisome-deficient Chinese hamster ovary cell mutant, ZPG208, of complementation group 17. The deduced primary sequence showed ∼25% amino acid identity with the yeast Pex3p, thereby we termed this cDNA ratPEX3 (RnPEX3). Human and Chinese hamster Pex3p showed 96 and 94% identity to rat Pex3p and had 373 amino acids. Pex3p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. A homozygous, inactivating missense mutation, G to A at position413, in a codon (GGA) for Gly138 and resulting in a codon (GAA) for Glu was the genetic cause of peroxisome deficiency of complementation group 17 ZPG208. The peroxisome-restoring activity apparently required the full length of Pex3p, whereas its N-terminal part from residues 1 to 40 was sufficient to target a fusion protein to peroxisomes. We also demonstrated that Pex3p binds the farnesylated peroxisomal membrane protein Pex19p. Moreover, upon expression of PEX3 in ZPG208, peroxisomal membrane vesicles were assembled before the import of soluble proteins such as PTS2-tagged green fluorescent protein. Thus, Pex3p assembles membrane vesicles before the matrix proteins are translocated.

scholarly journals Modulation ofN-Ethylmaleimide-sensitive Factor Activity upon Amino Acid Deprivation

2005 ◽  
Vol 280 (16) ◽  
pp. 16219-16226 ◽  
Author(s):  
Hagai Shorer ◽  
Nira Amar ◽  
Ari Meerson ◽  
Zvulun Elazar
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Yellow Fluorescent Protein ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Sensitive Factor ◽  
Vesicular Stomatitis

Adaptation of eukaryotic cells to changing environmental conditions entails rapid regulation of protein targeting and transport to specific organelles. Such adaptation is well exemplified in mammalian cells exposed to nitrogen starvation that are triggered to form and transport autophagosomes to lysosomes, thus constituting an inducible intracellular trafficking pathway. Here we investigated the relationship between the general secretory machinery and the autophagic pathway in Chinese hamster ovary cells grown in the absence of amino acid. Utilizing VSVG-YFP (vesicular stomatitis virus G protein fused to yellow fluorescent protein) and norepinephrine as markers for constitutive and regulated exocytosis, respectively, we found that secretion is attenuated in cells grown in media lacking amino acid. Such decrease in exocytosis stems from partial inhibition ofN-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells. These findings expose a novel cellular strategy to attenuate secretion of proteins under conditions of limited amino acid supply.

scholarly journals The importomer is a peroxisomal membrane protein translocase

2020 ◽  
Author(s):  
James S Martenson ◽  
Hanson Tam ◽  
Alexander J McQuown ◽  
Dvir Reif ◽  
Jing Zhou ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Matrix Protein ◽  
Protein Import ◽  
Protein Targeting ◽  
Fundamental Principle ◽  
Specific Protein ◽  
En Bloc ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein ◽  
Protein Biogenesis

ABSTRACTThree sites of membrane protein biogenesis (the endoplasmic reticulum, mitochondria and chloroplasts) receive unfolded substrates from organelle-specific protein targeting factors, then integrate them using separate translocation channels. Peroxisomes also receive membrane proteins from known targeting factors, but whether a separate translocase is needed for integration remains unknown. Here, using a novel genetic screening strategy, we reveal that the importomer–known for matrix protein import–integrates the peroxisomal membrane protein Pex14. In importomer mutants, Pex14 is arrested in a pre-integrated state on the peroxisome surface. To undergo integration, a Pex14 translocation signal binds the importomer’s substrate receptor Pex5 at a distinct site from matrix proteins. En bloc translocation of an engineered protein complex with Pex14’s luminal region argues that integration occurs without substrate unfolding. Our work shows that the handling of membrane protein targeting and integration by discrete machineries is a fundamental principle shared by diverse membrane protein biogenesis pathways.

scholarly journals Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p

2010 ◽  
Vol 29 (24) ◽  
pp. 4083-4093 ◽  
Author(s):  
Yasuhiko Sato ◽  
Hiroyuki Shibata ◽  
Toru Nakatsu ◽  
Hiroaki Nakano ◽  
Yoshinori Kashiwayama ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Structural Basis ◽  
Peroxisomal Membrane ◽  
Protein Carrier ◽  
Peroxisomal Membrane Protein

scholarly journals Allosteric modulation of peroxisomal membrane protein recognition by farnesylation of the peroxisomal import receptor PEX19

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Leonidas Emmanouilidis ◽  
Ulrike Schütz ◽  
Konstantinos Tripsianes ◽  
Tobias Madl ◽  
Juliane Radke ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Allosteric Modulation ◽  
Protein Recognition ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein ◽  
Import Receptor

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

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