Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p

Roslyn M. Bill, Kristina Hedfalk, Sara Karlgren, Jonathan G.L. Mullins, Jan Rydström, Stefan Hohmann

Research output: Contribution to journalArticle

Abstract

Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved. To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p. We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators.
Original languageEnglish
Pages (from-to)36543-36549
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number39
DOIs
Publication statusPublished - 28 Sep 2001

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Fungal Proteins
Yeast
Glycerol
Yeasts
Mutation
Mutant Proteins
Site-Directed Mutagenesis
Saccharomyces cerevisiae
Mutagenesis
Proteins
Pore structure
Escherichia coli
Cells

Bibliographical note

This research was originally published in the Journal of Biological Chemistry. Roslyn M. Bill, Kristina Hedfalk, Sara Karlgren, Jonathan G. L. Mullins, Jan Rydström and Stefan Hohmann. Analysis of the Pore of the Unusual Major Intrinsic Protein Channel, Yeast Fps1p. J. Biol. Chem. 2001, 276:36543-36549. © the American Society for Biochemistry and Molecular Biology

Keywords

  • Amino Acid Motifs
  • Aquaporins
  • Bacterial Outer Membrane Proteins
  • Cell Membrane
  • DNA Primers
  • Escherichia coli
  • Escherichia coli Proteins
  • Fungal Proteins
  • Glycerol
  • Immunoblotting
  • Membrane Proteins
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Plasmids
  • Point Mutation
  • Protein Binding
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Time Factors

Cite this

Bill, R. M., Hedfalk, K., Karlgren, S., Mullins, J. G. L., Rydström, J., & Hohmann, S. (2001). Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p. Journal of Biological Chemistry, 276(39), 36543-36549. https://doi.org/10.1074/jbc.M105045200
Bill, Roslyn M. ; Hedfalk, Kristina ; Karlgren, Sara ; Mullins, Jonathan G.L. ; Rydström, Jan ; Hohmann, Stefan. / Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 39. pp. 36543-36549.
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Bill, RM, Hedfalk, K, Karlgren, S, Mullins, JGL, Rydström, J & Hohmann, S 2001, 'Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p', Journal of Biological Chemistry, vol. 276, no. 39, pp. 36543-36549. https://doi.org/10.1074/jbc.M105045200

Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p. / Bill, Roslyn M.; Hedfalk, Kristina; Karlgren, Sara; Mullins, Jonathan G.L.; Rydström, Jan; Hohmann, Stefan.

In: Journal of Biological Chemistry, Vol. 276, No. 39, 28.09.2001, p. 36543-36549.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p

AU - Bill, Roslyn M.

AU - Hedfalk, Kristina

AU - Karlgren, Sara

AU - Mullins, Jonathan G.L.

AU - Rydström, Jan

AU - Hohmann, Stefan

N1 - This research was originally published in the Journal of Biological Chemistry. Roslyn M. Bill, Kristina Hedfalk, Sara Karlgren, Jonathan G. L. Mullins, Jan Rydström and Stefan Hohmann. Analysis of the Pore of the Unusual Major Intrinsic Protein Channel, Yeast Fps1p. J. Biol. Chem. 2001, 276:36543-36549. © the American Society for Biochemistry and Molecular Biology

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N2 - Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved. To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p. We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators.

AB - Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved. To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p. We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators.

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KW - Aquaporins

KW - Bacterial Outer Membrane Proteins

KW - Cell Membrane

KW - DNA Primers

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KW - Escherichia coli Proteins

KW - Fungal Proteins

KW - Glycerol

KW - Immunoblotting

KW - Membrane Proteins

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Mutation

KW - Plasmids

KW - Point Mutation

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - Saccharomyces cerevisiae

KW - Saccharomyces cerevisiae Proteins

KW - Time Factors

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DO - 10.1074/jbc.M105045200

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