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 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 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.

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.

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.

scholarly journals Import of stably folded proteins into peroxisomes.

1995 ◽  
Vol 6 (6) ◽  
pp. 675-683 ◽  
Author(s):  
P A Walton ◽  
P E Hill ◽  
S Subramani
Keyword(s):  
Colloidal Gold ◽  
Disulfide Bonds ◽  
Protein Unfolding ◽  
Matrix Proteins ◽  
Gold Particles ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Folded Proteins ◽  
Carboxy Terminal ◽  
Peroxisomal Targeting Signal

By virtue of their synthesis in the cytoplasm, proteins destined for import into peroxisomes are obliged to traverse the single membrane of this organelle. Because the targeting signal for most peroxisomal matrix proteins is a carboxy-terminal tripeptide sequence (SKL or its variants), these proteins must remain import competent until their translation is complete. We sought to determine whether stably folded proteins were substrates for peroxisomal import. Prefolded proteins stabilized with disulfide bonds and chemical cross-linkers were shown to be substrates for peroxisomal import, as were mature folded and disulfide-bonded IgG molecules containing the peroxisomal targeting signal. In addition, colloidal gold particles conjugated to proteins bearing the peroxisomal targeting signal were translocated into the peroxisomal matrix. These results support the concept that proteins may fold in the mammalian cytosol, before their import into the peroxisome, and that protein unfolding is not a prerequisite for peroxisomal import.

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