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Uptake Assay for Radiolabeled Peptides in Yeast

Authors: Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
, Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
, Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, New York, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
,  date: 11/20/2016, 1134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2026.

[Abstract] We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems....

Halo Assay for Toxic Peptides and Other Compounds in Microorganisms

Authors: Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
, Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
, Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
,  date: 11/20/2016, 1181 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2025.

[Abstract] We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may ...

Design and Functional Analysis of Fluorescent Nitrate and Peptide Transporter Activity Sensors in Yeast Cultures

Authors: Cheng-Hsun Ho
Cheng-Hsun HoAffiliation: Department of Plant Biology, Carnegie Science, Stanford, USA
Present address: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
For correspondence: zcybele3@sinica.edu.tw
Bio-protocol author page: a2900
 and Wolf B. Frommer
Wolf B. FrommerAffiliation: Department of Plant Biology, Carnegie Science, Stanford, USA
Bio-protocol author page: a1915
,  date: 2/5/2016, 1947 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1728.

[Abstract] This protocol describes the methods used to engineer and deploy genetically encoded fluorescence activity reporters for nitrate and peptide transporter activity in yeast cells. Fusion of the dual-affinity nitrate transceptor CHL1/AtNRT1.1/AtNPF6.3 or four different peptide transporters (AtPTR1, 2, 4, ...

Quantification of Extracellular Ammonium Concentrations and Transporter Activity in Yeast Using AmTrac Fluorescent Sensors

Authors: Cindy Ast
Cindy AstAffiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, USA
Bio-protocol author page: a1914
, Wolf B. Frommer
Wolf B. FrommerAffiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, USA
Bio-protocol author page: a1915
, Guido Grossmann
Guido GrossmannAffiliation: Cell Networks-Cluster of Excellence and Centre for Organismal Studies (COS) Heidelberg Bioresources, Universität Heidelberg, Heidelberg, Germany
Bio-protocol author page: a1916
 and Roberto De Michele
Roberto De MicheleAffiliation: Institute of Biosciences and Bioresources, National Research council of Italy (CNR-IBBR), Palermo, Italy
For correspondence: roberto.demichele@cnr.it
Bio-protocol author page: a1917
,  date: 1/5/2015, 3171 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1372.

[Abstract] AmTracs are the first example of “activity sensors”, since they report the activity of ammonium transporters by means of fluorescence readout in vivo (De Michele et al., 2013). AmTracs are based on a single fluorescent protein, a circularly permuted GFP (cpGFP), inserted into the cytosolic loop connecting ...

Synchronization of Saccharomyces cerevisiae Cells in G1 Phase of the Cell Cycle

Authors: Ildiko Unk
Ildiko UnkAffiliation: The Institute of Genetics, Biological Research Center of The Hungarian Academy of Sciences, Szeged, Hungary
For correspondence: unk.ildiko@brc.mta.hu
Bio-protocol author page: a1741
 and Andreea Daraba
Andreea DarabaAffiliation: The Institute of Genetics, Biological Research Center of The Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a1742
,  date: 10/20/2014, 4680 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1273.

[Abstract] The baker’s yeast, Saccharomyces cerevisiae is a widely used model organism in molecular biology because of the high homology it shares with human cells in many basic cellular processes such as DNA replication, repair, recombination, transcription, and because of the ease its genome can be manipulated. ...

Spot Assay for Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
,  date: 1/5/2012, 19524 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.16.

[Abstract] This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies....
1 

Spot Assay for Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
,  date: 1/5/2012, 19524 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.16.

[Abstract] This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies....

Synchronization of Saccharomyces cerevisiae Cells in G1 Phase of the Cell Cycle

Authors: Ildiko Unk
Ildiko UnkAffiliation: The Institute of Genetics, Biological Research Center of The Hungarian Academy of Sciences, Szeged, Hungary
For correspondence: unk.ildiko@brc.mta.hu
Bio-protocol author page: a1741
 and Andreea Daraba
Andreea DarabaAffiliation: The Institute of Genetics, Biological Research Center of The Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a1742
,  date: 10/20/2014, 4680 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1273.

[Abstract] The baker’s yeast, Saccharomyces cerevisiae is a widely used model organism in molecular biology because of the high homology it shares with human cells in many basic cellular processes such as DNA replication, repair, recombination, transcription, and because of the ease its genome can be manipulated. ...

Quantification of Extracellular Ammonium Concentrations and Transporter Activity in Yeast Using AmTrac Fluorescent Sensors

Authors: Cindy Ast
Cindy AstAffiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, USA
Bio-protocol author page: a1914
, Wolf B. Frommer
Wolf B. FrommerAffiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford, USA
Bio-protocol author page: a1915
, Guido Grossmann
Guido GrossmannAffiliation: Cell Networks-Cluster of Excellence and Centre for Organismal Studies (COS) Heidelberg Bioresources, Universität Heidelberg, Heidelberg, Germany
Bio-protocol author page: a1916
 and Roberto De Michele
Roberto De MicheleAffiliation: Institute of Biosciences and Bioresources, National Research council of Italy (CNR-IBBR), Palermo, Italy
For correspondence: roberto.demichele@cnr.it
Bio-protocol author page: a1917
,  date: 1/5/2015, 3171 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1372.

[Abstract] AmTracs are the first example of “activity sensors”, since they report the activity of ammonium transporters by means of fluorescence readout in vivo (De Michele et al., 2013). AmTracs are based on a single fluorescent protein, a circularly permuted GFP (cpGFP), inserted into the cytosolic loop connecting ...

Design and Functional Analysis of Fluorescent Nitrate and Peptide Transporter Activity Sensors in Yeast Cultures

Authors: Cheng-Hsun Ho
Cheng-Hsun HoAffiliation: Department of Plant Biology, Carnegie Science, Stanford, USA
Present address: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
For correspondence: zcybele3@sinica.edu.tw
Bio-protocol author page: a2900
 and Wolf B. Frommer
Wolf B. FrommerAffiliation: Department of Plant Biology, Carnegie Science, Stanford, USA
Bio-protocol author page: a1915
,  date: 2/5/2016, 1947 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1728.

[Abstract] This protocol describes the methods used to engineer and deploy genetically encoded fluorescence activity reporters for nitrate and peptide transporter activity in yeast cells. Fusion of the dual-affinity nitrate transceptor CHL1/AtNRT1.1/AtNPF6.3 or four different peptide transporters (AtPTR1, 2, 4, ...

Halo Assay for Toxic Peptides and Other Compounds in Microorganisms

Authors: Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
, Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
, Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
,  date: 11/20/2016, 1181 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2025.

[Abstract] We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may ...

Uptake Assay for Radiolabeled Peptides in Yeast

Authors: Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
, Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
, Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, New York, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
,  date: 11/20/2016, 1134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2026.

[Abstract] We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems....
1 
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