Extraction of cell surface-associated proteins from living yeast cells

Yeast ◽  
2007 ◽  
Vol 24 (4) ◽  
pp. 253-258 ◽  
Author(s):  
Frans M. Klis ◽  
Marian de Jong ◽  
Stanley Brul ◽  
Piet W. J. de Groot
Keyword(s):  
Cell Surface ◽  
Yeast Cells ◽  
Associated Proteins

Cell Surface-associated Proteins of Gastrointestinal Strains of Lactobacilli

1992 ◽  
Vol 5 (3) ◽  
Author(s):  
P. Chagnaud ◽  
H. F. Jenkinson ◽  
G. W. Tannock
Keyword(s):  
Cell Surface ◽  
Associated Proteins

scholarly journals Cell surface-associated proteins which bind native type IV collagen or gelatin.

1984 ◽  
Vol 259 (9) ◽  
pp. 5915-5922 ◽  
Author(s):  
M Kurkinen ◽  
A Taylor ◽  
J I Garrels ◽  
B L Hogan
Keyword(s):  
Cell Surface ◽  
Type Iv Collagen ◽  
Type Iv ◽  
Associated Proteins

scholarly journals Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport.

1992 ◽  
Vol 118 (2) ◽  
pp. 285-299 ◽  
Author(s):  
H Liu ◽  
A Bretscher
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Synthetic Lethality ◽  
Vesicular Transport ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Apparent Loss ◽  
Abnormal Accumulation ◽  
Late Step ◽  
Actin Cables

Disruption of the yeast tropomyosin gene TPM1 results in the apparent loss of actin cables from the cytoskeleton (Liu, H., and A. Bretscher. 1989. Cell. 57:233-242). Here we show that TPM1 disrupted cells grow slowly, show heterogeneity in cell size, have delocalized deposition of chitin, and mate poorly because of defects in both shmooing and cell fusion. The transit time of alpha-factor induced a-agglutinin secretion to the cell surface is longer than in isogenic wild-type strains, and some of the protein is mislocalized. Many of the TPM1-deleted cells contain abundant vesicles, similar in morphology to late secretory vesicles, but without an abnormal accumulation of intermediates in the delivery of either carboxypeptidase Y to the vacuole or invertase to the cell surface. Combinations of the TPM1 disruption with sec13 or sec18 mutations, which affect early steps in the secretory pathway, block vesicle accumulation, while combinations with sec1, sec4 or sec6 mutations, which affect a late step in the secretory pathway, have no effect on the vesicle accumulation. The phenotype of the TPM1 disrupted cells is very similar to that of a conditional mutation in the MYO2 gene, which encodes a myosin-like protein (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). The myo2-66 conditional mutation shows synthetic lethality with the TPM1 disruption, indicating that the MYO2 and TPM1 gene products may be involved in the same, or parallel function. We conclude that tropomyosin, and by inference actin cables, may facilitate directed vesicular transport of components to the correct location on the cell surface.

scholarly journals Cell patterning by secretion-induced plasma membrane flows

2020 ◽  
Author(s):  
Veneta Gerganova ◽  
Iker Lamas ◽  
David M. Rutkowski ◽  
Aleksandar Vještica ◽  
Daniela Gallo Castro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Negative Feedback ◽  
Reaction Diffusion ◽  
Cell Patterning ◽  
Yeast Cells ◽  
Membrane Insertion ◽  
Gtpase Activating Proteins ◽  
Associated Proteins ◽  
Bulk Membrane ◽  
Polarized Exocytosis

AbstractCells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modelling, optogenetics and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane-associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.One Sentence SummaryExocytosis causes bulk membrane flows that drag associated proteins and form a negative feedback restricting the exocytic site.

scholarly journals Rapid purification of glycosyl-phosphatidylinositol-anchored alkaline phosphatase from human neutrophils after up-regulation to the cell surface.

1993 ◽  
Vol 41 (9) ◽  
pp. 1367-1372 ◽  
Author(s):  
T J Cain ◽  
Y Liu ◽  
T Kobayashi ◽  
J M Robinson
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Surface ◽  
Human Neutrophils ◽  
Extracellular Medium ◽  
Isolation And Purification ◽  
Glycosyl Phosphatidylinositol ◽  
Associated Proteins ◽  
Membrane Bound ◽  
Pre Treatment ◽  
Rapid Purification

Alkaline phosphatase (APase) belongs to a growing family of membrane-associated proteins tethered to the lipid bilayer via a glycosyl-phosphatidylinositol (GPI) anchor. Human neutrophils contain an intracellular pool of APase associated with a novel membrane-bound compartment. Stimulation of neutrophils with the chemotactic peptide formyl-Met-Leu-Phe (fMLP) leads to rapid up-regulation of essentially all of the APase to sites in continuity with the extracellular medium. Pre-treatment of neutrophils with cytochalasin B (cyto B) followed by fMLP likewise leads to expression of the enzyme on the cell surface and a dramatic alteration in cell morphology, but subsequent internalization of the plasmalemma is minimized. Pre-treatment with cyto B and fMLP has been used for isolation and purification of neutrophil APase. Specifically, neutrophils were treated with phosphatidylinositol-specific phospholipase C to release GPI-anchored proteins from the cell surface. APase was purified from supernatants of these preparations by electrophoresis in a non-denaturing gel system and subsequent electroelution. With this approach we rapidly purified neutrophil APase to homogeneity; this protein was then used for immunization. Immunoblotting, ELISA, and immunocytochemical localization were used to characterize the resulting antibodies.

scholarly journals Cell Surface Engineering of a β-Galactosidase for Galactooligosaccharide Synthesis

2009 ◽  
Vol 75 (18) ◽  
pp. 5938-5942 ◽  
Author(s):  
Yumei Li ◽  
Lili Lu ◽  
Hongmei Wang ◽  
Xiaodong Xu ◽  
Min Xiao
Keyword(s):  
Cell Surface ◽  
Surface Engineering ◽  
Yeast Cells ◽  
Penicillium Expansum ◽  
Yeast Nitrogen Base ◽  
Gene Encoding ◽  
Nitrogen Base ◽  
Reading Frame ◽  
Acid Protein ◽  
Casamino Acids

ABSTRACT A novel gene encoding transglycosylating β-galactosidase (BGase) was cloned from Penicillium expansum F3. The sequence contained a 3,036-bp open reading frame encoding a 1,011-amino-acid protein. This gene was subsequently expressed on the cell surface of Saccharomyces cerevisiae EBY-100 by galactose induction. The BGase-anchored yeast could directly utilize lactose to produce galactooligosaccharide (GOS), as well as the by-products glucose and a small quantity of galactose. The glucose was consumed by the yeast, and the galactose was used for BGase expression, thus greatly facilitating GOS synthesis. The GOS yield reached 43.64% when the recombinant yeast was cultivated in yeast nitrogen base-Casamino Acids medium containing 100 g/liter initial lactose at 25°C for 5 days. The yeast cells were harvested and recycled for the next batch of GOS synthesis. During sequential operations, both oligosaccharide synthesis and BGase expression were maintained at high levels with GOS yields of over 40%, and approximately 8 U/ml of BGase was detected in each batch.

scholarly journals Intracellular vesicle clusters are organelles that synthesize extracellular vesicle–associated cargo proteins in yeast

2020 ◽  
Vol 295 (9) ◽  
pp. 2650-2663 ◽  
Author(s):  
Chelsea M. Winters ◽  
Ly Q. Hong-Brown ◽  
Hui-Ling Chiang
Keyword(s):  
Protein Synthesis ◽  
Cell Communication ◽  
Protein Translation ◽  
Extracellular Vesicle ◽  
Yeast Cells ◽  
Foreign Protein ◽  
Intact Cells ◽  
Intracellular Vesicle ◽  
Cargo Proteins ◽  
Associated Proteins

Extracellular vesicles (EVs) play important roles in cell-cell communication. In budding yeast (Saccharomyces cerevisiae), EVs function as carriers to transport cargo proteins into the periplasm for storage during glucose starvation. However, intracellular organelles that synthesize these EV-associated cargo proteins have not been identified. Here, we investigated whether cytoplasmic organelles—called intracellular vesicle clusters (IVCs)—serve as sites for the synthesis of proteins targeted for secretion as EV-associated proteins. Using proteomics, we identified 377 IVC-associated proteins in yeast cells grown under steady-state low-glucose conditions, with the largest group being involved in protein translation. Isolated IVCs exhibited protein synthesis activities that required initiation and elongation factors. We have also identified 431 newly synthesized proteins on isolated IVCs. Expression of 103Q-GFP, a foreign protein with a long polyglutamine extension, resulted in distribution of this protein as large puncta that co-localized with IVC markers, including fructose-1,6-bisphosphatase (FBPase) and the vacuole import and degradation protein Vid24p. We did not observe this pattern in cycloheximide-treated cells or in cells lacking VID genes, required for IVC formation. The induction of 103Q-GFP on IVCs adversely affected total protein synthesis in intact cells and on isolated IVCs. This expression also decreased levels of EV-associated cargo proteins in the extracellular fraction without affecting the number of secreted EVs. Our results provide important insights into the functions of IVCs as sites for the synthesis of EV-associated proteins targeted for secretion to the periplasm.

scholarly journals New Assay for Measuring Cell Surface Hydrophobicities of Candida dubliniensis andCandida albicans

2001 ◽  
Vol 8 (3) ◽  
pp. 585-587 ◽  
Author(s):  
M. A. Jabra-Rizk ◽  
W. A. Falkler ◽  
W. G. Merz ◽  
T. F. Meiller
Keyword(s):  
Cell Surface ◽  
Anaerobic Bacterium ◽  
Hydrophobic Interactions ◽  
Cell Surface Hydrophobicity ◽  
Fusobacterium Nucleatum ◽  
Surface Hydrophobicity ◽  
Yeast Cells ◽  
Pathogenic Yeast ◽  
Yeast Cell Surface ◽  
The Many

ABSTRACT Hydrophobic interactions, based on cell surface hydrophobicity (CSH), are among the many and varied mechanisms of adherence deployed by the pathogenic yeast Candida albicans. Recently it was shown that, unlike C. albicans, C. dubliniensisis a species that exhibits an outer fibrillar layer consistent with constant CSH. Previously, C. dubliniensis grown at 25 or 37°C was shown to coaggregate with the oral anaerobic bacteriumFusobacterium nucleatum. C. albicans, however, demonstrated similar coaggregation only when hydrophobic or grown at 25°C. This observation implied that coaggregation of Candida cells with F. nucleatum is associated with a hydrophobic yeast cell surface. To test this hypothesis, 42 C. albicans and 40 C. dubliniensis clinical isolates, including a C. albicans hydrophobic variant, were grown at 25 and 37°C and tested with the established hydrophobicity microsphere assay, which determines CSH levels based on the number of microspheres attached to the yeast cells. The coaggregation assay was performed in parallel experiments. All C. dubliniensis isolates grown at either temperature, hydrophobic 25°C-grown C. albicans isolates, and the C. albicans hydrophobic variant, unlike the 37°C-hydrophilic C. albicans isolates, exhibited hydrophobic CSH levels with the microsphere assay and simultaneously showed maximum, 4+, coaggregation with F. nucleatum. The parallel results obtained for C. dubliniensis using both assays support the use of the CoAg assay both as a rapid assay to determine CSH and to differentiate between C. dubliniensisand C. albicans.

scholarly journals Cell Surface-associated Proteins of Gastrointestinal Strains of Lactobacilli

1992 ◽  
Vol 5 (3) ◽  
pp. 121-131 ◽  
Author(s):  
P. Chagnaud ◽  
H. F. Jenkinson ◽  
G. W. Tannock
Keyword(s):  
Cell Surface ◽  
Associated Proteins
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