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.

scholarly journals Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins.

1988 ◽  
Vol 107 (3) ◽  
pp. 897-905 ◽  
Author(s):  
S J Gould ◽  
G A Keller ◽  
S Subramani
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Acid Oxidase ◽  
Carboxy Terminus ◽  
Amino Acid Oxidase ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Acyl Coa Oxidase ◽  
Peroxisomal Targeting Signal

As part of an effort to understand how proteins are imported into the peroxisome, we have sought to identify the peroxisomal targeting signals in four unrelated peroxisomal proteins: human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and rat acyl-CoA oxidase. Using gene fusion experiments, we have identified a region of each protein that can direct heterologous proteins to peroxisomes. In each case, the peroxisomal targeting signal is contained at or near the carboxy terminus of the protein. For catalase, the peroxisomal targeting signal is located within the COOH-terminal 27 amino acids of the protein. For hydratase:dehydrogenase, D-amino acid oxidase, and acyl-CoA oxidase, the targeting signals are located within the carboxy-terminal 15, 14, and 15 amino acids, respectively. A tripeptide of the sequence Ser-Lys/His-Leu is present in each of these targeting signals as well as in the peroxisomal targeting signal identified in firefly luciferase (Gould, S.J., G.-A. Keller, and S. Subramani. 1987. J. Cell Biol. 105:2923-2931). When the peroxisomal targeting signal of the hydratase:dehydrogenase is mutated so that the Ser-Lys-Leu tripeptide is converted to Ser-Asn-Leu, it can no longer direct proteins to peroxisomes. We suggest that this tripeptide is an essential element of at least one class of peroxisomal targeting signals.

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 Light and electron microscopic localization of D-aspartate oxidase in peroxisomes of bovine kidney and liver: an immunocytochemical study.

1996 ◽  
Vol 44 (9) ◽  
pp. 1013-1019 ◽  
Author(s):  
K Zaar
Keyword(s):  
Protein A ◽  
Cell Types ◽  
Subcellular Fractionation ◽  
Systematic Investigation ◽  
Kidney Cortex ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Peroxisomal Enzyme ◽  
Bovine Kidney

D-Aspartate oxidase (EC 1.4.3.1; D-ASPOX) specifically oxidizes the D-isomers of dicarboxylic amino acids such as aspartic or glutamic acid. Subcellular fractionation experiments in the past showed its association with peroxisome preparations in kidney cortex and liver. However, no information exists on the in situ localization and distribution of the enzyme in different cell types. We have purified the enzyme from the bovine kidney and raised an antibody against it in rabbits. The monospecificity of the antibody has been confirmed by Western blotting and it does not crossreact with D-amino, acid oxidase. Immunohistochemical localization of the antigen in bovine kidney and liver with the streptavidin-biotin-peroxidase technique revealed a punctate localization in the epithelial cells of proximal nephron tubules, particularly in the straight P-3 segment, as well as in hepatocytes. This is consistent with a localization in peroxisomes. Best results have been obtained with Carnoy-fixed material after paraffin embedding or after fixation with formaldehyde-glutaraldehyde in cryostat sections. Immunoelectron microscopy with protein A-gold confirms the peroxisomal localization of D-ASPOX. Gold particles are distributed over the matrix, suggestive of a peroxisomal matrix enzyme. This is the first report on the localization of D-ASPOX, a little-known peroxisomal enzyme. The techniques described and the antibody prepared will now allow systematic investigation of its tissue distribution.

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.

Metabolic control of peroxisome abundance

1999 ◽  
Vol 112 (10) ◽  
pp. 1579-1590 ◽  
Author(s):  
C.C. Chang ◽  
S. South ◽  
D. Warren ◽  
J. Jones ◽  
A.B. Moser ◽  
...  
Keyword(s):  
Metabolic Control ◽  
Matrix Protein ◽  
Protein Import ◽  
Zellweger Syndrome ◽  
Mutant Gene ◽  
Peroxisomal Membrane ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Complementation Groups ◽  
Peroxisomal Targeting Signal

Zellweger syndrome and related disorders represent a group of lethal, genetically heterogeneous diseases. These peroxisome biogenesis disorders (PBDs) are characterized by defective peroxisomal matrix protein import and comprise at least 10 complementation groups. The genes defective in seven of these groups and more than 90% of PBD patients are now known. Here we examine the distribution of peroxisomal membrane proteins in fibroblasts from PBD patients representing the seven complementation groups for which the mutant gene is known. Peroxisomes were detected in all PBD cells, indicating that the ability to form a minimal peroxisomal structure is not blocked in these mutants. We also observed that peroxisome abundance was reduced fivefold in PBD cells that are defective in the PEX1, PEX5, PEX12, PEX6, PEX10, and PEX2 genes. These cell lines all display a defect in the import of proteins with the type-1 peroxisomal targeting signal (PTS1). In contrast, peroxisome abundance was unaffected in cells that are mutated in PEX7 and are defective only in the import of proteins with the type-2 peroxisomal targeting signal. Interestingly, a fivefold reduction in peroxisome abundance was also observed for cells lacking either of two PTS1-targeted peroxisomal beta-oxidation enzymes, acyl-CoA oxidase and 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase. These results indicate that reduced peroxisome abundance in PBD cells may be caused by their inability to import these PTS1-containing enzymes. Furthermore, the fact that peroxisome abundance is influenced by peroxisomal 105-oxidation activities suggests that there may be metabolic control of peroxisome abundance.

Real time imaging reveals a peroxisomal reticulum in living cells

2000 ◽  
Vol 113 (20) ◽  
pp. 3663-3671 ◽  
Author(s):  
M. Schrader ◽  
S.J. King ◽  
T.A. Stroh ◽  
T.A. Schroer
Keyword(s):  
Real Time ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Real Time Imaging ◽  
Peroxisomal Targeting ◽  
Microtubule Motor ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.

scholarly journals Expression of the Cymbidium Ringspot Virus 33-Kilodalton Protein in Saccharomyces cerevisiae and Molecular Dissection of the Peroxisomal Targeting Signal

2004 ◽  
Vol 78 (9) ◽  
pp. 4744-4752 ◽  
Author(s):  
Beatriz Navarro ◽  
Luisa Rubino ◽  
Marcello Russo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Ringspot Virus ◽  
Intermediate Step ◽  
Cell Organelles ◽  
Peroxisomal Membrane ◽  
Essential Components ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

ABSTRACT Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.

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