Immunodetection of spectrin-like proteins in yeasts.
Spectrin, a component of the membrane skeleton in erythrocytes and other animal cells, has also been identified in plant and fungal cells. However, its postulated role, i.e., the maintenance of shape and elasticity of the plasma membrane, is probably not exerted in walled cells. To study spectrin in these cells, we chose yeasts because of a high morphological variability of their life cycle. The localization of spectrin in the cells and protoplasts of Saccharomyces cerevisiae and Schizosaccharomyces japonicus var. versatilis was detected by immunoblotting, indirect immunofluorescence, and immunogold electron microscopy techniques with the use of anti-chicken and anti-human erythrocyte spectrin antibodies. A protein band of 220-240 kDa and some bands of lower relative mass were detected in cell and protoplast extracts of both yeast strains. Spectrin-like proteins were revealed by fluorescence microscopy at cell surfaces and in vacuolar membranes. Immunogold-labelling showed spectrin-like proteins in the plasma membrane, endoplasmic reticulum, vacuoles, nuclei, vesicles, mitochondria, and cell walls. The topology of spectrin was not affected by actin depolymerization with Latrunculin B nor was it changed in either act1-1 or cdc42 mutants, under restrictive conditions. Under osmotic stress, both spectrin and actin were delocalized and appeared in the form of large clusters in the cytoplasm. It is concluded that a protein cross-reacting with spectrin antibodies is present in fission and budding yeasts. Generally, it is located in the proximity of the plasma membrane and other intracellular membranes, probably as a part of the membrane skeleton. No evidence of its relationship to either actin or growth zones of the cell can be provided.
Slaninová
Iva
I
Department of Biology, Faculty of Medicine, Masaryk University, Jostova 10, 66243 Brno, Czech Republic. ipokorna@med.muni.cz
Holubárová
Alena
A
Svoboda
Augustin
A
D000199:Actins / Q000737:chemistry; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002457:Cell Extracts / Q000276:immunology; D003470:Culture Media; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000032:analysis* / Q000276:immunology; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000648:ultrastructure; D009154:Mutation; D009997:Osmotic Pressure; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D013049:Spectrin / Q000032:analysis* / Q000276:immunology; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002457:Cell Extracts; D003470:Culture Media; D005656:Fungal Proteins; D008565:Membrane Proteins; D013844:Thiazoles; D053778:Thiazolidines; C041393:spectrin-like proteins; D013049:Spectrin; C037068:latrunculin B