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Title: A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

Abstract

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD bindingmore » domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39.« less

Authors:
; ;  [1]
  1. (Univ. of Illinois, Chicago (USA))
Publication Date:
OSTI Identifier:
6828350
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Cell Biology; (USA); Journal Volume: 110:4
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; MEMBRANE PROTEINS; MOLECULAR STRUCTURE; AUTORADIOGRAPHY; CELL MEMBRANES; CHEMICAL BONDS; ELECTRON MICROSCOPY; EUGLENA; IODINE ISOTOPES; MOLECULAR WEIGHT; PEPTIDE HYDROLASES; PROTEIN STRUCTURE; TRYPSIN; TWO-DIMENSIONAL ELECTROPHORESIS; ALGAE; ANIMALS; CELL CONSTITUENTS; ELECTROPHORESIS; ENZYMES; EUGLENOPHYCOTA; HYDROLASES; INVERTEBRATES; ISOTOPES; MASTIGOPHORA; MEMBRANES; MICROORGANISMS; MICROSCOPY; ORGANIC COMPOUNDS; PLANTS; PROTEINS; PROTOZOA; SERINE PROTEINASES; UNICELLULAR ALGAE 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Rosiere, T.K., Marrs, J.A., and Bouck, G.B. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis. United States: N. p., 1990. Web. doi:10.1083/jcb.110.4.1077.
Rosiere, T.K., Marrs, J.A., & Bouck, G.B. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis. United States. doi:10.1083/jcb.110.4.1077.
Rosiere, T.K., Marrs, J.A., and Bouck, G.B. 1990. "A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis". United States. doi:10.1083/jcb.110.4.1077.
@article{osti_6828350,
title = {A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis},
author = {Rosiere, T.K. and Marrs, J.A. and Bouck, G.B.},
abstractNote = {The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39.},
doi = {10.1083/jcb.110.4.1077},
journal = {Journal of Cell Biology; (USA)},
number = ,
volume = 110:4,
place = {United States},
year = 1990,
month = 4
}
  • This work is a preliminary characterization of two adapted Euglena gracilis cell lines, one to cadmium and the other to pentachlorophenol. Growth curve analysis indicate that tolerance to one pollutant did not protect against the second pollutant. These suggest that metabolic pathways that are induced by one pollutant are specific for this pollutant. This specificity is detectable at the level of gene expression. 16 refs., 6 figs.
  • The effect of x rays on the formation of chlorophyll, and on the activity of NADP-linked glyceraldehyde-3-phosphate dehydrogenase (GPD) was investigated in non-dividing, dark-grown colorless Euglena gracilis (strain Z), during exposure to light. The increase after 48 h of both chlorophyll and GPD activity was severely inhibited by x rays over a range 0 to 80 krad. The activity of the GPD already present was not inhibited by doses of up to 65 krad. Inhibitors of translation, actinomycin C at 134 gamma /ml, actinomycin D, 5- fluorouracil at 1.5 x 10/sup -2/ M, and nifampicin at 250 gamma /ml didmore » not significantly influence the synthesis of chlorophyll and GPD. (UK)« less
  • The physiochemical properties and physiological significance of the cadmium-binding protein (CdBP) of the algae Euglena gracilis have been studied. Following in vivo exposure of cells to 0.4 or 1.3 ..mu..g/mL of Cd/sup 2 +/, all the cytosolic Cd is bound to high molecular weight species. At 4.7 ..mu..g/mL, appreciable CdBP has formed in cells grown under illumination or in the dark. The large pool of very low molecular weight zinc species previously reported is increased when cells are exposed to high cadmium levels. Two distinct species, BP-1 and BP-2 are resolved by ion-exchange chromatography on DEAE-Sephadex. Unusually high conductivities aremore » required to displace them, indicating that they are very negatively charged proteins at pH 8.6. The pH for half-titration of bound Cd/sup 2 +/ is between 5 and 6. Neither form of the CdBP cross-reacts with antibodies to rat liver metallothionein (MT) antibodies. The structural, chemical, and functional differences between the Euglena CdBPs and mammalian MTs are discussed. When cells are exposed to high levels of Cu, a CuBP is induced, and the very low molecular weight zinc band is depleted.« less
  • Diflubenzuron (I) and its major degradation products 4-chlorophenyl urea (II), 2,6-difluorobenzoic acid (III) and 4-chloroaniline (IV) were tested for their activity on Euglena gracilis Z. The inhibition on the growth and on the incorporation of glycine-U-/sub 14/C in the protein of Euglena was measured in the presence of I-IV ranging 10 to 200 ppm. 4-chloroaniline caused a considerable inhibition at every tested level whereas I-III slightly affected only the incorporation. Therefore, it must be inferred that diflubenzuron shows no effect on growth and protein biosynthesis for this nontarget organism.