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.

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 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.

scholarly journals Two independent peroxisomal targeting signals in catalase A of Saccharomyces cerevisiae.

1993 ◽  
Vol 120 (3) ◽  
pp. 665-673 ◽  
Author(s):  
F Kragler ◽  
A Langeder ◽  
J Raupachova ◽  
M Binder ◽  
A Hartig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Citrate Synthase ◽  
Amino Terminal ◽  
New Variant ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Reporter Proteins ◽  
Carboxy Terminal ◽  
Peroxisomal Targeting Signal

In contrast to many other peroxisomal proteins catalase A contains at least two peroxisomal targeting signals each sufficient to direct reporter proteins to peroxisomes. One of them resides at the extreme carboxy terminus constituting a new variant of this signal, -SSNSKF, not active in monkey kidney cells (Gould, S. J., G. A. Keller, N. Hosken, J. Wilkinson, and S. Subramani 1989. J. Cell Biol. 108:1657-1664). However, this signal is completely dispensable for import of catalase A itself. In its amino-terminal third this protein contains another peroxisomal targeting signal sufficient to direct reporter proteins into microbodies. This internal signal depends on the context. The nature of this targeting signal might be a short defined sequence or a structural feature recognized by import factors. In addition, we have demonstrated that the carboxy-terminal seven amino acids of citrate synthase of Saccharomyces cerevisiae encoded by CIT2 and containing the canonical -SKL represents a targeting signal sufficient to direct reporter proteins to peroxisomes.

Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae

1992 ◽  
Vol 12 (12) ◽  
pp. 5593-5599
Author(s):  
K K Singh ◽  
G M Small ◽  
A S Lewin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Citrate Synthase ◽  
Signal Sequence ◽  
Carboxyl Terminus ◽  
Density Gradients ◽  
Mitochondrial Targeting ◽  
Amino Terminal ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.

scholarly journals Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (12) ◽  
pp. 5593-5599 ◽  
Author(s):  
K K Singh ◽  
G M Small ◽  
A S Lewin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Citrate Synthase ◽  
Signal Sequence ◽  
Carboxyl Terminus ◽  
Density Gradients ◽  
Mitochondrial Targeting ◽  
Amino Terminal ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.

scholarly journals Involvement of the Saccharomyces cerevisiae Hydrolase Ldh1p in Lipid Homeostasis

2011 ◽  
Vol 10 (6) ◽  
pp. 776-781 ◽  
Author(s):  
Mykhaylo O. Debelyy ◽  
Sven Thoms ◽  
Melanie Connerth ◽  
Günther Daum ◽  
Ralf Erdmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lipid Droplets ◽  
Functional Characterization ◽  
State Level ◽  
Lipid Homeostasis ◽  
Nonpolar Lipid ◽  
Triacylglycerol Lipase ◽  
Content Type ◽  
Nonpolar Lipids

ABSTRACTHere, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p. Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in the hydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of theSaccharomyces cerevisiaeΔldh1strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels.

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.

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.

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