Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

scholarly journals Antibodies directed against the peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins.

1990 ◽  
Vol 110 (1) ◽  
pp. 27-34 ◽  
Author(s):  
S J Gould ◽  
S Krisans ◽  
G A Keller ◽  
S Subramani
Keyword(s):  
Amino Acids ◽  
Western Blot ◽  
Rat Liver ◽  
Synthetic Peptide ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal ◽  
Peptide Antibodies

We have previously shown that the peroxisomal targeting signal in firefly luciferase consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine (Gould, S.J., G.A. Keller, N. Hosken, J. Wilkinson, and S. Subramani, 1989. J. Cell Biol. 108:1657-1664). Antibodies were raised against a synthetic peptide that contained this tripeptide at its COOH terminus. Immunofluorescence and immunocryoelectron microscopy revealed that the anti-peptide antibodies specifically detected peroxisomes in mammalian cells. Further characterization revealed that the antibodies were primarily directed against the COOH-terminal three amino acids of the peptide. In Western blot experiments, the antibodies recognized 15-20 rat liver peroxisomal proteins, but reacted with only a few proteins from other subcellular compartments. These results provide independent immunological evidence that the peroxisomal targeting signal identified in firefly luciferase is present in many peroxisomal proteins.

scholarly journals Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase.

1987 ◽  
Vol 105 (6) ◽  
pp. 2923-2931 ◽  
Author(s):  
S G Gould ◽  
G A Keller ◽  
S Subramani
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Carboxy Terminus ◽  
Peroxisomal Membrane ◽  
Amino Terminal ◽  
Peroxisomal Targeting ◽  
Firefly Luciferase Gene ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

Translocation of proteins across membranes of the endoplasmic reticulum, mitochondrion, and chloroplast has been shown to be mediated by targeting signals present in the transported proteins. To test whether the transport of proteins into peroxisomes is also mediated by a peptide targeting signal, we have studied the firefly luciferase gene that encodes a protein transported to peroxisomes in both insect and mammalian cells. We have identified two regions of luciferase which are necessary for transport of this protein into peroxisomes. We demonstrate that one of these, region II, represents a peroxisomal targeting signal because it is both necessary and sufficient for directing cytosolic proteins to peroxisomes. The signal is no more than twelve amino acids long and is located at the extreme carboxy-terminus of luciferase. The location of the targeting signal for translocation across the peroxisomal membrane therefore differs from the predominantly amino-terminal location of signals responsible for transport across the membranes of the endoplasmic reticulum, chloroplast, or mitochondrion.

Transport of microinjected proteins into peroxisomes of mammalian cells: inability of Zellweger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal

1992 ◽  
Vol 12 (2) ◽  
pp. 531-541
Author(s):  
P A Walton ◽  
S J Gould ◽  
J R Feramisco ◽  
S Subramani
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Zellweger Syndrome ◽  
Fibroblast Cell ◽  
Artificial Hybrid ◽  
Peroxisomal Targeting ◽  
Hybrid Substrates ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

Previous work has shown that the firefly (Photinus pyralis) luciferase contains a C-terminal peroxisomal targeting signal consisting of the tripeptide Ser-Lys-Leu. This report describes the microinjection of two proteins, (i) luciferase and (ii) albumin conjugated to a peptide ending in the sequence Ser-Lys-Leu, into mammalian cells grown in tissue culture. Following microinjection, incubation of the cells at 37 degrees C resulted in peroxisomal transport of these exogenous proteins into catalase-containing vesicles. The translocation was both time and temperature dependent. The transport could be inhibited by coinjection of synthetic peptides bearing various peroxisomal targeting signal motifs. These proteins could be transported into peroxisomes in normal human fibroblast cell lines but not in cell lines derived from patients with Zellweger syndrome. These results demonstrate that microinjection of peroxisomal proteins yields an authentic in vivo system with which to study peroxisomal transport. Furthermore, these results reveal that the process of peroxisomal transport does not involve irreversible modification of the protein, that artificial hybrid substrates can be transported and used as tools to study peroxisomal transport, and that the defect in Zellweger syndrome is indeed the inability to transport proteins containing the Ser-Lys-Leu targeting signal into the peroxisomal lumen.

scholarly journals A conserved tripeptide sorts proteins to peroxisomes.

1989 ◽  
Vol 108 (5) ◽  
pp. 1657-1664 ◽  
Author(s):  
S J Gould ◽  
G A Keller ◽  
N Hosken ◽  
J Wilkinson ◽  
S Subramani
Keyword(s):  
Protein Import ◽  
Firefly Luciferase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Luciferase Gene ◽  
Luciferase Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal ◽  
Made In

The firefly luciferase protein contains a peroxisomal targeting signal at its extreme COOH terminus (Gould et al., 1987). Site-directed mutagenesis of the luciferase gene reveals that this peroxisomal targeting signal consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine. When this tripeptide is appended to the COOH terminus of a cytosolic protein (chloramphenicol acetyltransferase), it is sufficient to direct the fusion protein into peroxisomes. Additional mutagenesis experiments reveal that only a limited number of conservative changes can be made in this tripeptide targeting signal without abolishing its activity. These results indicate that peroxisomal protein import, unlike other types of transmembrane translocation, is dependent upon a conserved amino acid sequence.

scholarly journals A novel dynein-type AAA+ protein with peroxisomal targeting signal type 2

2020 ◽  
Vol 167 (5) ◽  
pp. 429-432
Author(s):  
Tsuneo Imanaka ◽  
Kosuke Kawaguchi
Keyword(s):  
Mammalian Cells ◽  
Ring Structure ◽  
Family Protein ◽  
Peroxisomal Targeting ◽  
Signal Type ◽  
Targeting Signal ◽  
Aaa Protein ◽  
Peroxisomal Targeting Signal

Abstract Peroxisomal matrix proteins are imported into peroxisomes in a process mediated by peroxisomal targeting signal (PTS) type 1 and 2. The PTS2 proteins are imported into peroxisomes after binding with Pex7p. Niwa et al. (A newly isolated Pex7-binding, atypical PTS2 protein P7BP2 is a novel dynein-type AAA+ protein. J Biochem 2018;164:437–447) identified a novel Pex7p-binding protein in CHO cells and characterized the subcellular distribution and molecular properties of the human homologue, ‘P7BP2’. Interestingly, P7BP2 possesses PTS2 at the NH2 terminal and six putative AAA+ domains. Another group has suggested that the protein also possesses mitochondrial targeting signal at the NH2 terminal. In fact, the P7BP2 expressed in mammalian cells is targeted to both peroxisomes and mitochondria. The purified protein from Sf9 cells is a monomer and has a disc-like ring structure, suggesting that P7BP2 is a novel dynein-type AAA+ family protein. The protein expressed in insect cells exhibits ATPase activity. P7BP2 localizes to peroxisomes and mitochondria, and has a common function related to dynein-type ATPases in both organelles.

scholarly journals Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: involvement of the peroxisomal targeting signal.

1992 ◽  
Vol 118 (3) ◽  
pp. 499-508 ◽  
Author(s):  
P A Walton ◽  
S J Gould ◽  
R A Rachubinski ◽  
S Subramani ◽  
J R Feramisco
Keyword(s):  
Pichia Pastoris ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Protein Transport ◽  
Zellweger Syndrome ◽  
Alcohol Oxidase ◽  
Peroxisomal Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

This report describes the microinjection of a purified peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures. Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments. The transport was time and temperature dependent. Vesicles containing alcohol oxidase could be detected as long as 6 d after injection. Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide peroxisomal targeting signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined. Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes. While the inhibition of transport of alcohol oxidase by peptides containing the peroxisomal targeting signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria. Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH peroxisomal targeting signal into peroxisomes (Walton et al., 1992). The implications of this result for the mechanism of peroxisomal protein transport are discussed.

scholarly journals The Putative Saccharomyces cerevisiae Hydrolase Ldh1p Is Localized to Lipid Droplets

2011 ◽  
Vol 10 (6) ◽  
pp. 770-775 ◽  
Author(s):  
Sven Thoms ◽  
Mykhaylo O. Debelyy ◽  
Melanie Connerth ◽  
Günther Daum ◽  
Ralf Erdmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Subcellular Localization ◽  
Lipid Droplets ◽  
Peroxisomal Enzyme ◽  
Content Type ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Nonpolar Lipids ◽  
Peroxisomal Targeting Signal ◽  
Hydrolase Family

ABSTRACT Here, we report the identification of a novel hydrolase in Saccharomyces cerevisiae . Ldh1p (systematic name, Ybr204cp) comprises the typical GXSXG-type lipase motif of members of the α/β-hydrolase family and shares some features with the peroxisomal lipase Lpx1p. Both proteins carry a putative peroxisomal targeting signal type1 (PTS1) and can be aligned with two regions of homology. While Lpx1p is known as a peroxisomal enzyme, subcellular localization studies revealed that Ldh1p is predominantly localized to lipid droplets, the storage compartment of nonpolar lipids. Ldh1p is not required for the function and biogenesis of peroxisomes, and targeting of Ldh1p to lipid droplets occurs independently of the PTS1 receptor Pex5p.

Analyses in transfected cells and in vitro of a putative peroxisomal targeting signal of rat liver serine:pyruvate aminotransferase

2002 ◽  
Vol 118 (4) ◽  
pp. 321-328 ◽  
Author(s):  
Takuji Mizuno ◽  
Kouichi Ito ◽  
Chiharu Uchida ◽  
Masatoshi Kitagawa ◽  
Arata Ichiyama ◽  
...  
Keyword(s):  
Rat Liver ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

scholarly journals cDNA cloning and expression of the flavoprotein d-aspartate oxidase from bovine kidney cortex

1997 ◽  
Vol 322 (3) ◽  
pp. 729-735 ◽  
Author(s):  
Tatjana SIMONIC ◽  
Stefano DUGA ◽  
Armando NEGRI ◽  
Gabriella TEDESCHI ◽  
Massimo MALCOVATI ◽  
...  
Keyword(s):  
Active Enzyme ◽  
Cloning And Expression ◽  
Kidney Cortex ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Peroxisomal Enzyme ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
First Time ◽  
Peroxisomal Targeting Signal

The isolation and sequencing of the complete cDNA coding for a d-aspartate oxidase, as well as the overexpression of the recombinant active enzyme, are reported for the first time. This 2022 bp cDNA, beside the coding portion, comprises a 5´ untranslated tract and the whole 3´ region including the polyadenylation signal and the poly(A) tail. The encoded protein comprises 341 amino acids, with the last three residues (-Ser-Lys-Leu) representing a peroxisomal targeting signal 1 (PTS1), hitherto unknown for this protein. The overexpression of recombinant d-aspartate oxidase was achieved in a prokaryotic system, and a soluble and active enzyme was obtained which accounted for about 10% of total bacterial protein. Comparisons with the known cDNAs for mammalian d-amino acid oxidase, another peroxisomal enzyme, are also made. The close structural and functional similarities shared by these enzymes at the protein level are not reflected at the nucleic acid level.

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