Search results for "Intracellular Membranes"

Study of surface carbohydrates on isolated Golgi subfractions by fluorescent-lectin binding and flow cytometry

1995

The Golgi complex is a functionally heterogeneous subcellular structure that plays a key role in the synthesis, maturation, and sorting of newly synthesized glycoproteins. Fluorescent lectins have been used extensively to analyze surface glycoproteins by flow cytometry in whole cells and more recently in isolated subcellular organelles, such as mitochondria and chloroplasts. We report here the use of several fluorescein-isothiocyanate-conjugated lectins to detect and quantify specific surface sugars by flow cytometry on isolated elements from purified cis and trans-Golgi fractions from rat liver. Our results show that this approach may be useful to study Golgi composition and function, sinc…

Permeabilization of the erythrocyte membrane with streptolysin O allows access to the vacuolar membrane of Plasmodium falciparum and a molecular anal…

1997

Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O

1996

Plasmodium falciparum is an intracellular parasite of human red blood cells (RBCs). Like many other intracellular parasites, P. falciparum resides and develops within a parasitophorous vacuole which is bound by a membrane that separates the host cell cytoplasm from the parasite surface. Some parasite proteins are secreted into the vacuolar space and others are secreted, by an as yet poorly defined pathway, into the RBC cytosol. The transport of proteins from the parasite has been followed mainly using morphological methods. In search of an experimental system that would allow (i) dissection of the individual steps involved in transport from the parasite surface into the RBC cytosol, and (ii…

Organelle pH studies using targeted avidin and fluorescein–biotin

2000

Abstract Background: Mammalian organelles of the secretory pathway are of differing pH. The pH values form a decreasing gradient: the endoplasmic reticulum (ER) is nearly neutral, the Golgi is mildly acidic and the secretory granules are more acidic still (∼pH 5). The mechanisms that regulate pH in these organelles are still unknown. Results: Using a novel method, we tested whether differences in H + ‘leak' and/or counterion conductances contributed to the pH difference between two secretory pathway organelles. A pH-sensitive, membrane-permeable fluorescein–biotin was targeted to endoplasmic-reticulum- and Golgi-localized avidin-chimera proteins in HeLa cells. In live, intact cells, ER pH (…

Impaired Transporter Associated with Antigen Processing (TAP) Function Attributable to a Single Amino Acid Alteration in the Peptide TAP Subunit TAP1

2003

Abstract The heterodimeric peptide transporter TAP belongs to the ABC transporter family. Sequence comparisons with the P-glycoprotein and cystic fibrosis transmembrane conductance regulator and the functional properties of selective amino acids in these ABC transporters postulated that the glutamic acid at position 263 and the phenylalanine at position 265 of the TAP1 subunit could affect peptide transporter function. To define the role of both amino acids, TAP1 mutants containing a deletion or a substitution to alanine at position 263 or 265 were generated and stably expressed in murine and human TAP1−/− cells. The different TAP1 mutants were characterized in terms of expression and funct…

Liver microsomal membrane fluidity and microsomal desaturase activities in adult spontaneously hypertensive rats.

1997

OBJECTIVE The purpose of the present study was to investigate liver microsomal membrane fluidity simultaneously with membrane fatty acid composition and desaturase activities in spontaneously hypertensive rats (SHR). DESIGN AND METHODS The membrane fluidity was determined, after electron spin resonance (ESR) measurement, in SHR compared with normotensive Wistar-Kyoto (WKY) rats, by calculating the order parameter S from ESR spectra of 5-nitroxide stearate and 10-nitroxide stearate, used as spin-labelled fatty acids. Desaturase activities were measured by incubating SHR and WKY rat liver microsomes with [14C]-radiolabeled fatty acids as substrates for desaturation reactions. The fatty acid c…

The yeast osmosensitive mutant fps1Δ transformed by the cauliflower BobTIP1;1 aquaporin withstand a hypo-osmotic shock

2005

AbstractOsmoregulation plays an important role in cellular responses to osmotic stress in plants and in yeast. Aquaporins contribute to osmotic adjustment by facilitating transport of water or solutes across membranes. The tonoplastic water channel BobTIP1;1 (original name BobTIP26-1) genes are upregulated during dessication stress in cauliflower meristematic tissue. To investigate the physiological importance of BobTIP1;1, we expressed it in a Saccharomyces cerevisiae osmosensitive mutant fps1Δ. We showed that the defect in the yeast glycerol plasma membrane transporter is complemented by a plant cDNA encoding the aquaporin BobTIP1;1 which is localized in the vacuolar membrane of the compl…

The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its…

1996

Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786–5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been …

Different conformations of nascent polypeptides during translocation across the ER membrane

2000

Abstract Background In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated α-helices) may be possible within the ribosome. Results We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transfer…

Membrane-insertion fragments of Bcl-xL, Bax, and Bid.

2004

Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria and the endoplasmic reticulum, where they perform their function. The activity of these proteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm, probably through the formation of ion and/or protein transport channels. Most of these proteins contain a C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic alpha helices (alpha5-alpha6) similar to the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here, we report on the membrane-insertion properties of different segments from antiapopt…