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1.
Eur J Biochem ; 271(5): 1035-45, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15009215

ABSTRACT

Loss of functional emerin, a nuclear membrane protein, causes X-linked recessive Emery-Dreifuss muscular dystrophy. In a yeast two-hybrid screen, we found that emerin interacts with Btf, a death-promoting transcriptional repressor, which is expressed at high levels in skeletal muscle. Biochemical analysis showed that emerin binds Btf with an equilibrium affinity (KD) of 100 nm. Using a collection of 21 clustered alanine-substitution mutations in emerin, the residues required for binding to Btf mapped to two regions of emerin that flank its lamin-binding domain. Two disease-causing mutations in emerin, S54F and Delta95-99, disrupted binding to Btf. The Delta95-99 mutation was relatively uninformative, as this mutation also disrupts emerin binding to lamin A and a different transcription repressor named germ cell-less (GCL). In striking contrast, emerin mutant S54F, which binds normally to barrier-to-autointegration factor, lamin A and GCL, selectively disrupted emerin binding to Btf. We localized endogenous Btf in HeLa cells by indirect immunoflurorescence using affinity-purified antibodies against Btf. In nonapoptotic HeLa cells Btf was found in dot-like structures throughout the nuclear interior. However, within 3 h after treating cells with Fas antibody to induce apoptosis, the distribution of Btf changed, and Btf concentrated in a distinct zone near the nuclear envelope. These results suggest that Btf localization is regulated by apoptotic signals, and that loss of emerin binding to Btf may be relevant to muscle wasting in Emery-Dreifuss muscular dystrophy.


Subject(s)
DNA-Binding Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Muscular Dystrophy, Emery-Dreifuss/genetics , Muscular Dystrophy, Emery-Dreifuss/metabolism , Mutation, Missense , Repressor Proteins/metabolism , Thymopoietins/genetics , Thymopoietins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Apoptosis/physiology , DNA-Binding Proteins/genetics , HeLa Cells , Humans , Nuclear Envelope/metabolism , Nuclear Proteins , Protein Binding , Protein Structure, Tertiary , Repressor Proteins/genetics , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Two-Hybrid System Techniques
2.
Genes Cells ; 7(9): 881-7, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12296819

ABSTRACT

The spectral resolution of fluorescence microscope images in living cells is achieved by using a confocal laser scanning microscope equipped with grating optics. This capability of temporal and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP).


Subject(s)
Fluorescent Dyes/metabolism , Microscopy, Fluorescence/methods , Fluorescein-5-isothiocyanate/metabolism , Fluorescence Resonance Energy Transfer , Green Fluorescent Proteins , HeLa Cells , Humans , Luminescent Proteins/metabolism , Microscopy, Confocal , Recombinant Fusion Proteins/metabolism
3.
Mol Biol Cell ; 13(2): 711-22, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11854424

ABSTRACT

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.


Subject(s)
ATP-Binding Cassette Transporters , Adenosine Triphosphatases/genetics , Peroxisomes/physiology , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/deficiency , Animals , CHO Cells , Cricetinae , Fibroblasts/physiology , Fibroblasts/ultrastructure , Genetic Complementation Test , Humans , Membrane Proteins/physiology , Peroxisomes/ultrastructure
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