Secretion and Surface Display of Green Fluorescent Protein Using the Yeast Saccharomyces cerevisiae

2008 ◽  
Vol 21 (2) ◽  
pp. 349-357 ◽  
Author(s):  
Dagang Huang ◽  
Eric V. Shusta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase

2003 ◽  
Vol 371 (2) ◽  
pp. 533-540 ◽  
Author(s):  
Shary N. SHELTON ◽  
Barbara BARYLKO ◽  
Derk D. BINNS ◽  
Bruce F. HORAZDOVSKY ◽  
Joseph P. ALBANESI ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Distinct Type ◽  
Type Ii ◽  
Yeast Saccharomyces Cerevisiae ◽  
Molar Ratios ◽  
Ionic Detergent ◽  
Km Value ◽  
Green Fluorescent ◽  
Vacuolar Membranes

The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100min−1 were obtained at the PI/Triton X-100 ratio of 1:5. The Km value for ATP was 266μM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p–green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.

scholarly journals Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

2004 ◽  
Vol 70 (2) ◽  
pp. 961-966 ◽  
Author(s):  
Antje Eiden-Plach ◽  
Tatjana Zagorc ◽  
Tanja Heintel ◽  
Yvonne Carius ◽  
Frank Breinig ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Schizosaccharomyces Pombe ◽  
Protein Secretion ◽  
Candida Glabrata ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Foreign Protein ◽  
Green Fluorescent

ABSTRACT Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.

Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae

2001 ◽  
Vol 12 (9) ◽  
pp. 2614-2628 ◽  
Author(s):  
Yunrui Du ◽  
Marc Pypaert ◽  
Peter Novick ◽  
Susan Ferro-Novick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bifunctional Protein ◽  
Daughter Cells ◽  
Bud Neck ◽  
J Domain ◽  
Green Fluorescent ◽  
Cortical Endoplasmic Reticulum

In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found at the periphery of the cell and around the nucleus. The segregation of ER through the mother-bud neck may occur by more than one mechanism because perinuclear, but not peripheral ER, requires microtubules for this event. To identify genes whose products are required for cortical ER inheritance, we have used a Tn3-based transposon library to mutagenize cells expressing a green fluorescent protein-tagged ER marker protein (Hmg1p). This approach has revealed that AUX1/SWA2plays a role in ER inheritance. The COOH terminus of Aux1p/Swa2p contains a J-domain that is highly related to the J-domain of auxilin, which stimulates the uncoating of clathrin-coated vesicles. Deletion of the J-domain of Aux1p/Swa2p leads to vacuole fragmentation and membrane accumulation but does not affect the migration of peripheral ER into daughter cells. These findings suggest that Aux1p/Swa2p may be a bifunctional protein with roles in membrane traffic and cortical ER inheritance. In support of this hypothesis, we find that Aux1p/Swa2p localizes to ER membranes.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

scholarly journals Green Fluorescent Protein-Dal80p Illuminates up to 16 Distinct Foci That Colocalize with and Exhibit the Same Behavior as Chromosomal DNA Proceeding through the Cell Cycle of Saccharomyces cerevisiae

2001 ◽  
Vol 183 (15) ◽  
pp. 4636-4642 ◽  
Author(s):  
MacKenzie Distler ◽  
Ajit Kulkarni ◽  
Rajendra Rai ◽  
Terrance G. Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Leucine Zipper ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Live Cells ◽  
Nitrogen Catabolite Repression ◽  
Chromosomal Dna ◽  
Green Fluorescent

ABSTRACT Four GATA family DNA binding proteins mediate nitrogen catabolite repression-sensitive transcription in Saccharomyces cerevisiae. Gln3p and Gat1p are transcriptional activators, while Dal80p and Deh1p repress Gln3p- and Gat1p-mediated transcription by competing with these activators for binding to DNA. Strong Dal80p binding to DNA is thought to result from C-terminal leucine zipper-mediated dimerization. Many Dal80p binding site-homologous sequences are relatively evenly distributed across the S. cerevisiae genome, raising the possibility that Dal80p might be able to “stain” DNA. We demonstrate that cells containing enhanced green fluorescent protein-Dal80p (EGFP-Dal80p) exhibit up to 16 fluorescent foci that colocalize with DAPI (4′,6′-diamidino-2-phenylindole)-positive material and follow DNA movement through the cell cycle, suggesting that EGFP-Dal80p may indeed be useful for monitoring yeast chromosomes in live cells and in real time.

scholarly journals A Rapid Method to Determine the Stress Status of Saccharomyces cerevisiae by Monitoring the Expression of a Hsp12:Green Fluorescent Protein (GFP) Construct under the Control of the Hsp12 Promoter

2005 ◽  
Vol 10 (3) ◽  
pp. 253-259 ◽  
Author(s):  
Robert J. Karreman ◽  
George G. Lindsey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Stress ◽  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Fusion Protein ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Time Dependent ◽  
Gfp Fluorescence ◽  
Green Fluorescent

The gene for the green fluorescent protein (GFP) was fused in-frame to the 3′ end of HSP12. This construct was regulated by the HSP12 promoter in a pYES2 yeast expression vector. No fluorescence was observed in yeast growing exponentially in glucose-containing medium, but fluorescence was observed when the yeast entered the stationary phase. Fluorescence microscopy indicated that the fusion protein was localized to the peripheral regions of the cell as well as to the cytoplasm and the tonoplast. Subjecting the yeast to a variety of stresses known to induce HSP12 transcription, including salt, osmotic, ethanol, and heat stress, resulted in a time-dependent increase in GFP fluorescence. The use of this system as a method to assess the general stress status of yeast growing in an industrial application is proposed.

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