A regulatory domain in the C-terminal extension of the yeast glycerol channel Fps1p.
The Saccharomyces cerevisiae gene FPS1 encodes an aquaglyceroporin of the major intrinsic protein (MIP) family. The main function of Fps1p seems to be the efflux of glycerol in the adaptation of the yeast cell to lower external osmolarity. Fps1p is an atypical member of the family, because the protein is much larger (669 amino acids) than most MIPs due to long hydrophilic extensions in both termini. We have shown previously that a short domain in the N-terminal extension of the protein is required for restricting glycerol transport through the channel (Tamás, M. J., Karlgren, S., Bill, R. M., Hedfalk, K., Allegri, L., Ferreira, M., Thevelein, J. M., Rydström, J., Mullins, J. G. L., and Hohmann, S. (2003) J. Biol. Chem. 278, 6337-6345). Deletion of the N-terminal domain results in an unregulated channel, loss of glycerol, and osmosensitivity. In this work we have investigated the role of the Fps1p C terminus (139 amino acids). A set of eight truncations has been constructed and tested in vivo in a yeast fps1Delta strain. We have performed growth tests, membrane localization following cell fractionation, and glycerol accumulation measurements as well as an investigation of the osmotic stress response. Our results show that the C-terminal extension is also involved in restricting transport through Fps1p. We have identified a sequence of 12 amino acids, residues 535-546, close to the sixth transmembrane domain. This element seems to be important for controlling Fps1p function. Similar to the N-terminal domain, the C-terminal domain is amphiphilic and has a potential to dip into the membrane.
Hedfalk
Kristina
K
Department of Cell and Molecular Biology/Microbiology, Götegorg University, Götegorg, Sweden. kristina.hedfalk@molbiotech.chalmers.se
Bill
Roslyn M
RM
Mullins
Jonathan G L
JG
Karlgren
Sara
S
Filipsson
Caroline
C
Bergstrom
Johanna
J
Tamás
Markus J
MJ
Rydström
Jan
J
Hohmann
Stefan
S
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D017353:Gene Deletion; D005990:Glycerol / Q000737:chemistry; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009995:Osmosis; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors
C093263:FPS1 protein, S cerevisiae; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D005990:Glycerol