Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p.
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.
Bill
R M
RM
Department of Cell and Molecular Biology/Microbiology, Göteborg University, S-40530 Göteborg, Sweden.
Hedfalk
K
K
Karlgren
S
S
Mullins
J G
JG
Rydström
J
J
Hohmann
S
S
D020816:Amino Acid Motifs; D020346:Aquaporins; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000737:chemistry*; D005990:Glycerol / Q000378:metabolism; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors
D020346:Aquaporins; D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C093263:FPS1 protein, S cerevisiae; D005656:Fungal Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C078043:GlpF protein, E coli; D005990:Glycerol