Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

scholarly journals Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus.

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91 ◽  
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

scholarly journals C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal

1998 ◽  
Vol 336 (2) ◽  
pp. 367-371 ◽  
Author(s):  
Leen AMERY ◽  
Chantal BREES ◽  
Myriam BAES ◽  
Chiaki SETOYAMA ◽  
Retsu MIURA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
C Terminus ◽  
Targeting Signal ◽  
Fluorescent Pattern ◽  
Green Fluorescent ◽  
Positively Charged

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

scholarly journals Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal

2014 ◽  
Vol 13 (4) ◽  
pp. 539-547 ◽  
Author(s):  
VaNae Hamilton ◽  
Ujjal K. Singha ◽  
Joseph T. Smith ◽  
Ebony Weems ◽  
Minu Chaudhuri
Keyword(s):  
Amino Acid ◽  
Alternative Oxidase ◽  
Heterologous Protein ◽  
Amino Acid Residues ◽  
Mitochondrial Targeting ◽  
Content Type ◽  
Targeting Signal ◽  
Internal Signal ◽  
Trypanosome Alternative Oxidase

ABSTRACTRecognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form ofTrypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondriain vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to aT. bruceimitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.

The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo

1994 ◽  
Vol 14 (2) ◽  
pp. 1459-1464
Author(s):  
Y Minami ◽  
Y Kimura ◽  
H Kawasaki ◽  
K Suzuki ◽  
I Yahara
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Full Length ◽  
Yeast Cells ◽  
Amino Acid Residues ◽  
Haploid Strain ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hsp90 Alpha

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.

scholarly journals The PEST-like sequence of I kappa B alpha is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers.

1995 ◽  
Vol 15 (2) ◽  
pp. 872-882 ◽  
Author(s):  
M K Ernst ◽  
L L Dunn ◽  
N R Rice
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Interaction ◽  
Terminal Region ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Functional Capabilities ◽  
C Terminus

In most cells, proteins belonging to the Rel/NF-kappa B family of transcription factors are held in inactive form in the cytoplasm by an inhibitor protein, I kappa B alpha. Stimulation of the cells leads to degradation of the inhibitor and transit of active DNA-binding Rel/NF-kappa B dimers to the nucleus. I kappa B alpha is also able to inhibit DNA binding by Rel/NF-kappa B dimers in vitro, suggesting that it may perform the same function in cells when the activating signal is no longer present. Structurally, the human I kappa B alpha molecule can be divided into three sections: a 70-amino-acid N terminus with no known function, a 205-residue midsection composed of six ankyrin-like repeats, and a very acidic 42-amino-acid C terminus that resembles a PEST sequence. In this study we examined how the structural elements of the I kappa B alpha protein correlate with its functional capabilities both in vitro and in vivo. Using a battery of I kappa B alpha mutants, we show that (i) a dimer binds a single I kappa B alpha molecule, (ii) the acidic C-terminal region of I kappa B alpha is not required for protein-protein binding and does not mask the nuclear localization signal of the dimer, (iii) the same C-terminal region is required for inhibition of DNA binding, and (iv) this inhibition may be accomplished by direct interaction between the PEST-like region and the DNA-binding region of one of the subunits of the dimer.

scholarly journals The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo.

1994 ◽  
Vol 14 (2) ◽  
pp. 1459-1464 ◽  
Author(s):  
Y Minami ◽  
Y Kimura ◽  
H Kawasaki ◽  
K Suzuki ◽  
I Yahara
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Full Length ◽  
Yeast Cells ◽  
Amino Acid Residues ◽  
Haploid Strain ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hsp90 Alpha

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.

scholarly journals Acyl-Coenzyme A Dehydrogenases Are Localized on GLUT4-Containing Vesicles via Association with Insulin-Regulated Aminopeptidase in a Manner Dependent on Its Dileucine Motif

2002 ◽  
Vol 16 (5) ◽  
pp. 1049-1059 ◽  
Author(s):  
Hideki Katagiri ◽  
Tomoichiro Asano ◽  
Tetsuya Yamada ◽  
Toshifumi Aoyama ◽  
Yasushi Fukushima ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coenzyme A ◽  
Amino Acid Residues ◽  
Glutathione S Transferase ◽  
Medium Chain ◽  
Dileucine Motif ◽  
Chain Acyl ◽  
Acyl Coenzyme A ◽  
Long Chain

Abstract Insulin-regulated aminopeptidase (IRAP, also termed vp165) is known to be localized on the GLUT4-containing vesicles and to be recruited to the plasma membrane after stimulation with insulin. The cytoplasmic region of IRAP contains two dileucine motifs and acidic regions, one of which (amino acid residues 55–82) is reportedly involved in retention of GLUT4-containing vesicles. The region of IRAP fused with glutathione-S-transferase [GST-IRAP(55–82)] was incubated with lysates from 3T3-L1 adipocytes, leading to identification of long-chain, medium-chain, and short-chain acyl-coenzyme A dehydrogenases (ACDs) as the proteins associated with IRAP. The association was nearly abolished by mutation of the dileucine motif of IRAP. Immunoblotting of fractions prepared from sucrose gradient ultracentrifugation and vesicles immunopurified with anti-GLUT4 antibody revealed these ACDs to be localized on GLUT4-containing vesicles. Furthermore, 3-mercaptopropionic acid and hexanoyl-CoA, inhibitors of long-chain and medium-chain ACDs, respectively, induced dissociation of long-chain acyl-coenzyme A dehydrogenase and/or medium-chain acyl-coenzyme A dehydrogenase from IRAP in vitro as well as recruitment of GLUT4 to the plasma membrane and stimulation of glucose transport activity in permeabilized 3T3-L1 adipocytes. These findings suggest that ACDs are localized on GLUT4-containing vesicles via association with IRAP in a manner dependent on its dileucine motif and play a role in retention of GLUT4-containing vesicles to an intracellular compartment.

scholarly journals Influence of N-terminal acetylation and C-terminal proteolysis on the analgesic activity of β-endorphin

1980 ◽  
Vol 189 (3) ◽  
pp. 501-506 ◽  
Author(s):  
J F W Deakin ◽  
J O Doströvsky ◽  
D G Smyth
Keyword(s):  
Amino Acid ◽  
Opiate Receptors ◽  
Acetyl Derivative ◽  
Amino Acid Residues ◽  
Duration Of Action ◽  
Beta Endorphin ◽  
C Terminus ◽  
N Terminus ◽  
Specific Affinity

Removal of one, two and four amino-acid residues from the C-terminus of beta-endorphin (‘lipotropin C-Fragment’, lipotropin residues 61–91) led to the formation of peptides with progressively decreased analgesic potency; there was no change in the persistence of the analgesic effects. The four C-terminal residues are thus important for the activity of beta-endorphin, but not for the duration of action. Removal of eight amino-acid residues from the N-terminus provided a peptide that had no specific affinity for brain opiate receptors in vitro and was devoid of analgesic properties. The N-terminal sequence of beta-endorphin is therefore necessary for the production of analgesia, whereas the C-terminal residues confer potency. The N alpha-acetyl form of beta-endorphin had no specific affinity for brain opiate receptors in vitro and possessed no significant analgesic properties. Since lipotropin C'-Fragment (lipotropin residues 61–87) and the N alpha-acetyl derivative of beta-endorphin occur naturally in brain and pituitary and are only weakly active or inactive as opiates, it is suggested that proteolysis at the C-terminus and acetylation of the N-terminus of beta-endorphin may constitute physiological mechanisms for inactivation of this potent analgesic peptide.

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