Turnover of protein components of the plasma membrane of Saccharomyces cerevisiae

Herrero, Enrique; Pastor, Javier; Sentandreau, Rafael

doi:10.1016/0005-2736(82)90186-9

Cited by 5 publications

(9 citation statements)

References 24 publications

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“…Action ofZymolyase on whole cells. Doubly labelled cells were treated with Zymolyase (in the same proportion as for isolated walls) in an osmotically stabilized medium at 28 "C as described by Pastor et al (1982), except that the pretreatment step was omitted and treatment with the enzyme was carried out in the presence of 1 mM-PMSF. All solutions contained sodium azide at 2 m~ (final concentration).…”

Section: Methodsmentioning

confidence: 99%

“…In our previous work (Pastor et al, 1982), wall purification involved treatment of the walls with sodium deoxycholate. This step was omitted in the above experiments.…”

Section: Solubilization Of Wall Mannoproteins From S Cerevisiaementioning

confidence: 99%

“…In the latter case, incorporation of the 14C isotope was stopped by addition of an excess of a mixture of unlabelled amino acids (Pastor et al, 1982). The walls corresponding to 15 mg cells were used in each digestion.…”

Section: Solubilization Of Newly Incorporated and Older Mannoprotein mentioning

confidence: 99%

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Solubilization and Analysis of Mannoprotein Molecules from the Cell Wall of Saccharomyces cerevisiae

et al. 1984

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Purified walls from Saccharomyces cerevisiae were treated chemically to release intrinsic mannoproteins. Boiling in 2% SDS gave the best results, although treatment in 6 M-urea at room temperature also released significant amounts of mannoprotein radioactivity. Triton X-100, sodium deoxycholate and EDTA were poor solubilizers. Electrophoretic patterns of SDSor urea-released mannoproteins in SDS-acrylamide gels indicated a great heterogeneity of molecular species, with more than 60 bands. Zymolyase, a glucan-digesting complex, released about half of the mannoproteins, but these species showed an altered mobility on SDSacrylamide gels and had a lowered capacity for precipitation by ethanol. Action of the enzyme on isolated walls was favoured by dithiothreitol, as is the case with whole cells, and repeated treatments with SDS and Zymolyase released all of the mannoproteins from the wall. Solubilizing treatments other than SDS had a differential effect on recently or formerly incorporated mannoproteins in the wall. The results suggest an asymmetrical arrangement of molecules in the envelope and point to dynamic changes inside the wall as it thickens as a result of cell aging.Abbreviation : PMSF, phenylmethylsulphonyl fluoride.

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Section: Methodsmentioning

confidence: 99%

“…In our previous work (Pastor et al, 1982), wall purification involved treatment of the walls with sodium deoxycholate. This step was omitted in the above experiments.…”

Section: Solubilization Of Wall Mannoproteins From S Cerevisiaementioning

confidence: 99%

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Solubilization and Analysis of Mannoprotein Molecules from the Cell Wall of Saccharomyces cerevisiae

et al. 1984

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“…Samples of protein (80 pg) were run in PAGE as described previously (Herrero et al, 1982). In experiments with labelled proteins, constant radioactivity was loaded for each sample.…”

Section: Introductionmentioning

confidence: 99%

Wall Mannoproteins of the Yeast and Mycelial Cells of Candida albicuns: Nature of the Glycosidic Bonds and Polydispersity of Their Mannan Moieties

Elorza

Marcilla²,

Sentandreu³

1988

Microbiology

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Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6 % glucose) and 7 :< protein. Over 85 % of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked 0-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2.5 kDa N-glycosidically linked sugar chain and a 3 1.5 kDa protein moiety. Polydispersity in the high molecular mass mannoproteins was due to the N-linked sugar chains (mannan) with a molecular mass between 500 kDa and 20 kDa (average 100 kDa) in Y cells and between 400 kDa and 20 kDa (average 50 kDa) in M cells.Three mannoproteins of 34,30 and 29 kDa secreted by protoplasts were associated with the high molecular mass mannoproteins, suggesting that this type of interaction might be related to the regeneration of the cell wall.

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“…Key terms: Flow cytometry, Epstein-Barr virus (EBV), surface receptors, membrane protein turnover, fluoresceinated virus Recent approaches to the study of membrane protein turnover have involved detecting specific proteins by the time-consuming techniques of sodium dodecyl sulfate (SDS)-gel electrophoresis (5), immunoprecipitation (l), purification of individual proteins (201, and the use of fluorescent probes and fluorescence microscopy (2). Only the latter technique permits following the fate of a n individul membrane protein on single cells, but it is qualitative and insensitive.…”

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confidence: 99%

Application of flow cytometry to studies on the distribution, persistence, and regeneration rate of Epstein‐Barr virus receptors

McCune

Volsky

1984

Cytometry

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Flow cytometry has been applied to study the persistence and regeneration rate of Epstein-Barr virus (EBV) receptors and other membrane proteins in normal and tumor cells. EBV receptors were detected in a binding assay utilizing fluorescein isothiocyanate-conjugated virions (FITC-EBV). After stripping receptors from the cell surface with trypsin, binding of FITC-EBV was rapidly regenerated in the lymphoma cell line Loukes, reaching 75% of the control levels by 3 h. Fresh human lymphocytes regenerated receptors at a much slower rate, reaching 50% of control levels by 24 h. The regenerating receptors did not reappear simultaneously on all the cells. Subpopulation of new receptor-positive cells increased gradually during the incubation at 37°C. Reappearance of receptors was inhibited in the absence of protein synthesis. Conversely, receptors persisted in the nontrypsinized cells in the absence of protein synthesis. These results show that certain parameters of membrane receptor turnover, such as the change in receptor distribution within a cell population, receptor persistance, and regeneration rate, can be measured at the single-cell level by flow cytometry.Key terms: Flow cytometry, Epstein-Barr virus (EBV), surface receptors, membrane protein turnover, fluoresceinated virus Recent approaches to the study of membrane protein turnover have involved detecting specific proteins by the time-consuming techniques of sodium dodecyl sulfate (SDS)-gel electrophoresis (5), immunoprecipitation (l), purification of individual proteins (201, and the use of fluorescent probes and fluorescence microscopy (2). Only the latter technique permits following the fate of a n individul membrane protein on single cells, but it is qualitative and insensitive. Flow cytometry offers a rapid, sensitive, and quantitative method of detecting cell surface markers at the single cell level (10). Here we describe flow cytometry as a convenient and effective tool for the study of two aspects of membrane protein turnover: the persistence of membrane receptors in the presence and absence of protein synthesis inhibitor and regeneration of membrane receptors after stripping with trypsin. We have chosen to investigate membrane receptors for Epstein-Barr virus (EBV-R) because of our interest in the host cell range and biological activity of this human tumor agent. EBV-R have been demonstrated only on human and certain other primate B lymphocytes (6,181, strengthening the association between the virus and Burkitt's lymphoma (9). However, viral genome and nuclear antigen, EBNA, have also been regularly found in nasopharyngeal carcinoma cells (8,131. This indicates that under certain pathologic conditions EBV can bind to and penetrate into human epithelial cells. Moreover, while EBV-infected lymphocytes are normally recognized and eliminated by the immune system (41, the BL and NPC cells can successfully avert immune response (14), presumably as a result of some unknown malignancy-related cell surface changes. We have compared the persistence and regenerat...

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Turnover of protein components of the plasma membrane of Saccharomyces cerevisiae

Cited by 5 publications

References 24 publications

Solubilization and Analysis of Mannoprotein Molecules from the Cell Wall of Saccharomyces cerevisiae

Solubilization and Analysis of Mannoprotein Molecules from the Cell Wall of Saccharomyces cerevisiae

Wall Mannoproteins of the Yeast and Mycelial Cells of Candida albicuns: Nature of the Glycosidic Bonds and Polydispersity of Their Mannan Moieties

Application of flow cytometry to studies on the distribution, persistence, and regeneration rate of Epstein‐Barr virus receptors

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