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1.
Neuropharmacology ; 43(4): 482-91, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12367595

ABSTRACT

GABA(A) receptors are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two alpha, two beta and one gamma subunit that assemble around an aqueous pore and form an intrinsic chloride ion channel. Using full-length or truncated chimeric subunits it was demonstrated that homologous sequences from different subunit classes, alpha(1)(54-68), beta(3)(52-66), and gamma(2)(67-81), are important for assembly of GABA(A) receptors composed of alpha(1), beta(3), and gamma(2) subunits. In addition, evidence was provided that these sequences all are located in topologically homologous regions of the different subunits. Finally, it was demonstrated that the sequences investigated cause a selective assembly with certain subunits only and thus influence subunit arrangement within GABA(A) receptors.


Subject(s)
Receptors, GABA-A/biosynthesis , Amino Acid Sequence , Blotting, Western , Cells, Cultured , DNA, Complementary , Humans , Immunochemistry , Kidney/metabolism , Membrane Proteins/chemistry , Molecular Sequence Data , Precipitin Tests , Receptors, GABA-A/chemistry , Recombinant Fusion Proteins , Transfection
2.
Mol Genet Genomics ; 265(2): 276-86, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11361338

ABSTRACT

In the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype. Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.


Subject(s)
Peptides/physiology , Peroxisomes/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Animals , Biological Transport , Caenorhabditis elegans/genetics , Eukaryotic Cells , Genes, Reporter , Green Fluorescent Proteins , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Oleic Acid/metabolism , Peptides/genetics , Peroxisome-Targeting Signal 1 Receptor , Plants, Toxic , Receptors, Cytoplasmic and Nuclear/genetics , Repetitive Sequences, Amino Acid , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Nicotiana/genetics , Two-Hybrid System Techniques
3.
Mol Cell Biol Res Commun ; 4(2): 81-9, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11170837

ABSTRACT

The role of Saccharomyces cerevisiae Adr1p was examined with respect to the transcriptional regulation of the SPS19 gene encoding the peroxisomal beta-oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase. The SPS19 promoter contains both an oleate response element that binds the Pip2p-Oaf1p transcription factor as well as a canonical Adr1p-binding element, termed UAS1(SPS19). Northern analysis demonstrated that transcriptional up-regulation of SPS19 was abolished in cells devoid of Adr1p. Expression of an SPS19-lacZ reporter gene was shown to be quiescent in the adr1Delta mutant and abnormally elevated in cells containing multiple ADR1 copies. UAS1(SPS19) was able to compete for formation of a specific complex between recombinant Adr1p-LacZ and UAS1(CTA1) representing the corresponding Adr1p-binding element in the promoter of the catalase A gene, and to interact directly with this fusion protein. We conclude that in the presence of fatty acids in the medium transcription of SPS19 is directly regulated by both Pip2p-Oaf1p and Adr1p.


Subject(s)
DNA-Binding Proteins/pharmacology , Fatty Acid Desaturases/metabolism , Fungal Proteins/metabolism , Oleic Acid/metabolism , Oxidoreductases Acting on CH-CH Group Donors , Peroxisomes/enzymology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/physiology , Transcription Factors/metabolism , Transcription Factors/pharmacology , Binding Sites , Blotting, Northern , DNA Primers/chemistry , Electrophoresis, Agar Gel , Fatty Acid Desaturases/genetics , Gene Expression Regulation, Fungal/drug effects , Genetic Vectors , Lac Operon/physiology , Peroxisomes/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Spores, Fungal , Transcription Factors/genetics , Transcriptional Activation , Up-Regulation , beta-Galactosidase/metabolism
4.
Biochem J ; 344 Pt 3: 903-14, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10585880

ABSTRACT

Human 2,4-dienoyl-CoA reductase (2,4-reductase; DECR) and rat monofunctional Delta(3)-Delta(2)-enoyl-CoA isomerase (rat 3, 2-isomerase; ECI) are thought to be mitochondrial auxiliary enzymes involved in the beta-oxidation of unsaturated fatty acids. However, their function during this process has not been demonstrated. Although they lack obvious peroxisomal targeting signals (PTSs), both proteins have been suggested previously to also occur in the mammalian peroxisomal compartment. The putative function and peroxisomal location of the two mammalian proteins can be examined in yeast, since beta-oxidation of unsaturated fatty acids is a compartmentalized process in Saccharomyces cerevisiae requiring peroxisomal 2,4-dienoyl-CoA reductase (Sps19p) and peroxisomal 3, 2-isomerase (Eci1p). A yeast sps19Delta mutant expressing human 2, 4-reductase ending with the native C-terminus could not grow on petroselinic acid [cis-C(18:1(6))] medium but could grow when the protein was extended with a PTS tripeptide, SKL (Ser-Lys-Leu). We therefore reason that the human protein is a physiological 2, 4-reductase but that it is probably not peroxisomal. Rat 3, 2-isomerase expressed in a yeast eci1Delta strain was able to re-establish growth on oleic acid [cis-C(18:1(9))] medium irrespective of an SKL extension. Since we had shown that Delta(2,4) double bonds could not be metabolized extra-peroxisomally to restore growth of the sps19Delta strain, we postulate that rat 3,2-isomerase acted on the Delta(3) unsaturated metabolite of oleic acid by replacing the mutant's missing activity from within the peroxisomes. Immunoblotting of fractionated yeast cells expressing rat 3, 2-isomerase in combination with electron microscopy supported our proposal that the protein functioned in peroxisomes. The results presented here shed new light on the function and location of human mitochondrial 2,4-reductase and rat monofunctional 3,2-isomerase.


Subject(s)
Carbon-Carbon Double Bond Isomerases/metabolism , Fatty Acid Desaturases/metabolism , Fatty Acids, Unsaturated/metabolism , Mitochondria, Liver/enzymology , Oxidoreductases Acting on CH-CH Group Donors , Saccharomyces cerevisiae/enzymology , Animals , Cell Division , Dodecenoyl-CoA Isomerase , Gene Expression Regulation, Enzymologic , Humans , Microscopy, Electron , Mutation , Oleic Acid/metabolism , Oleic Acids/metabolism , Oligopeptides/genetics , Peroxisomes/enzymology , Plasmids , Rats , Saccharomyces cerevisiae/genetics
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