Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes.
The glycerol facilitator of Escherichia coli, GlpF, is a putative nonselective transport channel in the inner membrane of this Gram-negative bacterium. It is a member of the major intrinsic protein (MIP) family of transmembrane channel proteins. Its characterization has been hampered by the lack of a heterologous test system in which its activity can be examined in the absence of other bacterial proteins. Transport of glycerol mediated by this protein was characterized following injection of glpF mRNA into Xenopus laevis oocytes. The properties of GlpF were compared with those of the homologous plant water channel protein, gamma tonoplast intrinsic protein (gamma TIP), as well as the nonhomologous Xenopus K+ channel, Xsha. GlpF selectively transported glycerol but not water or ions, while gamma TIP and Xsha were specific for water and K+, respectively. Voltage clamp experiments showed that GlpF was not voltage-activated for ion transport. Glycerol transport via GlpF proved to be nonsaturable up to 200 mM and exhibited a low temperature of activation (Ea = 4.5 kcal/mol), consistent with the conclusion of Heller et al. (Heller, K. B., Lin, E. C. C., and Wilson, T. H. (1980) J. Bacteriol. 144, 274-278) that GlpF mediates glycerol diffusion via a pore type mechanism. GlpF-mediated transport of glycerol was blocked by mercuric ions (Hg2+) but not N-ethylmaleimide. The inhibitory effect of Hg2+ was partially prevented by inclusion of a high concentration of glycerol and reversed by mercaptoethanol. The results serve to characterize the transport properties of the E. coli glycerol facilitator.
Maurel
C
C
Department of Biology, University of California at San Diego, La Jolla 92093-0116.
Reizer
J
J
Schroeder
J I
JI
Chrispeels
M J
MJ
Saier
M H
MH
D000818:Animals; D020346:Aquaporins; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005260:Female; D005990:Glycerol / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism / Q000502:physiology*; D015221:Potassium Channels / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013816:Thermodynamics; D014982:Xenopus laevis
D020346:Aquaporins; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D015221:Potassium Channels; D012333:RNA, Messenger; C078043:GlpF protein, E coli; D005990:Glycerol