Glycerol facilitator of Escherichia coli: cloning of glpF and identification of the glpF product.

The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.     

Kinetics of glycerol uptake by the perfused rat liver. Membrane transport, phosphorylation and effect on NAD redox level.

The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular phosphorylation and competitive inhibition of glycerol phosphorylation by L-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K-m of the glycerol phosphorylation was 10 muM and the K-i of the L-glycerol 3-phosphate inhibition was 50 muM. The maximum activity (V) was 3.70 mumoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K-i was found to be somewhat lower in the intact organ. At low glycerol concentrations, a steep concentration gradient exists across the liver cell membrane. The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of L-glycerol 3-phosphate, not to the rate of glycerol uptake.     

Diffusion of glycerol through Escherichia coli aquaglyceroporin GlpF

The glycerol uptake facilitator, GlpF, a major intrinsic protein found in Escherichia coli, selectively conducts water and glycerol across the inner membrane. The free energy landscape characterizing the assisted transport of glycerol by this homotetrameric aquaglyceroporin has been explored by means of equilibrium molecular dynamics over a timescale spanning 0.12 μs. To overcome the free energy barriers of the conduction pathway, an adaptive biasing force is applied to the glycerol molecule confined in each of the four channels. The results illuminate the critical role played by intramolecular relaxation on the diffusion properties of the permeant. These free energy calculations reveal that glycerol tumbles and isomerizes on a timescale comparable to that spanned by its adaptive-biasing-force-assisted conduction in GlpF. As a result, reorientation and conformational equilibrium of glycerol in GlpF constitute a bottleneck in the molecular simulations of the permeation event. A profile characterizing the position-dependent diffusion of the permeant has been determined, allowing reaction rate theory to be applied for investigating conduction kinetics based on the measured free energy landscape.     

Physiological studies on regulation of glycerol utilization by the phosphoenolpyruvate:sugar phosphotransferase system in Enterococcus faecalis.

In vitro studies with purified glycerol kinase from Enterococcus faecalis have established that this enzyme is activated by phosphorylation of a histidyl residue in the protein, catalyzed by the phosphoenolpyruvate-dependent phosphotransferase system (PTS), but the physiological significance of this observation is not known. In the present study, the regulation of glycerol uptake was examined in a wild-type strain of E. faecalis as well as in tight and leaky ptsI mutants, altered with respect to their levels of enzyme I of the PTS. Glycerol kinase was shown to be weakly repressible by lactose and strongly repressible by glucose in the wild-type strain. Greatly reduced levels of glycerol kinase activity were also observed in the ptsI mutants. Uptake of glycerol into intact wild-type and mutant cells paralleled the glycerol kinase activities in extracts. Glycerol uptake in the leaky ptsI mutant was hypersensitive to inhibition by low concentrations of 2-deoxyglucose or glucose even though the rates and extent of 2-deoxyglucose uptake were greatly reduced. These observations provide strong support for the involvement of reversible PTS-mediated phosphorylation of glycerol kinase in the regulation of glycerol uptake in response to the presence or absence of a sugar substrate of the PTS in the medium. Glucose and 2-deoxyglucose were shown to elicit rapid efflux of cytoplasmic [14C]lactate derived from [14C]glycerol. This phenomenon was distinct from the inhibition of glycerol uptake and was due to phosphorylation of the incoming sugar by cytoplasmic phosphoenolpyruvate. Lactate appeared to be generated by sequential dephosphorylation and reduction of cytoplasmic phosphoenolpyruvate present in high concentrations in resting cells. The relevance of these findings to regulatory phenomena in other bacteria is discussed.     

Secondary structure and oligomerization of the E. coli glycerol facilitator

The Major Intrinsic Proteins are found throughout the bacterial, plant, and animal kingdoms and are responsible for the rapid transport of water and other small, polar solutes across membranes. The superfamily includes the aquaporins, the aquaglyceroporins, and the glycerol facilitators. We have overexpressed and purified the Escherichia coli inner membrane glycerol facilitator. Approximately 7.5 mg of 95% pure protein is obtained from 1 L of Escherichia coli cells using immobilized metal affinity chromatography. Well-resolved matrix-assisted laser desorption ionization mass spectra were obtained by solubilization of the protein in octyl-beta-D-glucopyranoside (M(r) = 33 650.3; error approximately 0.4%). The recombinant glycerol facilitator is inserted into the bacterial inner membrane, is functional, and is inhibited by HgCl(2). Polyacrylamide gel electrophoresis suggests that the facilitator is predominantly monomeric when solubilized with dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, and sodium dodecyl sulfate, but that it self-associates, forming soluble oligomers when urea is used during extraction. Similar oligomeric species are demonstrated to exist in the bacterial membrane by chemical cross-linking experiments. Circular dichroism analysis shows that the protein is predominantly alpha-helical. Helix content is significantly higher in protein prepared in the absence of urea (42-55%) than in its presence (32%). A possible role for the facilitator oligomers in interactions with, and regulation of, the glycerol kinase is discussed.     

Kinetics of glycerol uptake by the perfused rat liver Membrane transport, phosphorylation and effect on NAD redox level

The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular phosphorylation and competitive inhibition of glycerol phosphorylation by l-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K_m of the glycerol phosphorylation was was 10 μM and the K_i of the l-glycerol 3-phosphate inhibition was 50 μM. The maximum activity (V) was 3.70 μmoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K_i was found to be somewhat lower in the intact organ. At low glycerol concentrations, gradient exists across the liver cell membrane.The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of l-glycerol 3-phosphate, not to the rate of glycerol uptake.     

Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides. The coupling between transport and phosphorylation in inside-out vesicles

The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the literature. We have tested the coupling by analysing the kinetics of fructose phosphorylation by inside-out vesicles of Rhodopseudomonas sphaeroides. We conclude that fructose indeed has to enter the vesicle before it can be phosphorylated and therefore cannot be phosphorylated from the cytoplasmic side of the membrane. The Km of the phosphorylation reaction is 8 microM. The diffusion of fructose into the vesicle is a reaction that is also catalysed by the components of the phosphotransferase system. The undirectional flux from the cytoplasmic side of the membrane to the periplasmic side is a slow process with a Km of 4 mM and is rate-limiting over a large external fructose concentration range. In summary there is no phosphorylation without transport, but there is transport without phosphorylation.     

Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress.

The Saccharomyces cerevisiae FPS1 gene, which encodes a channel protein belonging to the MIP family, has been isolated previously as a multicopy suppressor of the growth defect of the fdp1 mutant (allelic to GGS1/TPS1) on fermentable sugars. Here we show that overexpression of FPS1 enhances glycerol production. Enhanced glycerol production caused by overexpression of GPD1 encoding glycerol-3-phosphate dehydrogenase also suppressed the growth defect of ggs1/tps1 delta mutants, suggesting a novel role for glycerol production in the control of glycolysis. The suppression of ggs1/tps1 delta mutants by GPD1 depends on the presence of Fps1. Mutants lacking Fps1 accumulate a greater part of the glycerol intracellularly, indicating that Fps1 is involved in glycerol efflux. Glycerol-uptake experiments showed that the permeability of the yeast plasma membrane for glycerol consists of an Fps1-independent component probably due to simple diffusion and of an Fps1-dependent component representing facilitated diffusion. The Escherichia coli glycerol facilitator expressed in a yeast fps1 delta mutant can restore the characteristics of glycerol uptake, production and distribution fully, but restores only partially growth of a ggs1/tps1 delta fps1 delta double mutant on glucose. Fps1 appears to be closed under hyperosmotic stress when survival depends on intracellular accumulation of glycerol and apparently opens rapidly when osmostress is lifted. The osmostress-induced High Osmolarity Glycerol (HOG) response pathway is not required for inactivation of Fps1. We conclude that Fps1 is a regulated yeast glycerol facilitator controlling glycerol production and cytosolic concentration, and might have additional functions.     

Promoter-Like Mutant with Increased Expression of the Glycerol Kinase Operon of Escherichia coli

A glycerol-specific phenotypic revertant isolated from a mutant of Escherichia coli missing enzyme I of the phosphoenolpyruvate phosphotransferase system was studied. This revertant is capable of producing higher levels of glycerol kinase and the protein mediating the facilitated diffusion of glycerol (facilitator) than wild-type cells. The kinase of the revertant is indistinguishable from the wild-type enzyme with respect to its sensitivity to feedback inhibition by fructose-1,6-diphosphate, its pH optimum, and its turnover number. The synthesis of glycerol kinase in strains bearing the suppressor locus is resistant to catabolite repression. The suppressor mutation mapped at the known glpK locus. Thus, it is suggested that the mutation occurred in the promoter of the operon specifying the kinase and the facilitator.     

Glucose uptake by Trypanosoma brucei. Rate-limiting steps in glycolysis and regulation of the glycolytic flux

Glucose uptake and metabolism in the bloodstream form of the glycosome-containing protozoan parasite Trypanosoma brucei was measured using 14C-labeled glucose in combination with the silicone oil centrifugation technique in short term (5-60 s) incubations. Glucose rather than glucose analogues was used to study the interrelation between the uptake process and the subsequent metabolic steps. Glucose uptake over the plasma membrane occurred by facilitated diffusion, which limited the overall glycolytic rate at external glucose concentrations (glcout) below 5 mM. At higher glcout another step, either transport over the glycosomal membrane or phosphorylation by hexokinase became rate-limiting. Mathematical modeling assuming that glucose uptake occurs by facilitated diffusion followed by an enzymatic step accurately predicts the experimental data. As predicted by the model, the internal concentration of non-metabolized glucose remains low till glcout = 5 mM and increases at higher external concentrations. In contrast to glucose, glycerol entered the cell by simple diffusion. Externally supplied glycerol did not affect glucose metabolism but externally added glucose interfered with glycerol metabolism in a way that suggests that the rate-limiting step is at the level of glycerol kinase. Our observations suggest that the bloodstream form of T. brucei adapts its glucose transport in a way that gives maximum yield at minimum expense.     

Transport and phosphorylation of choline in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

When sycamore cells were suspended in basal medium containing choline, the latter was taken up by the cells very rapidly. A facilitated diffusion system appertained at low concentrations of choline and exhibited Michaelis-Menten kinetics. At higher choline concentrations simple diffusion appeared to be the principal mode of uptake. Addition of choline to the perfusate of compressed sycamore cells monitored by 31P NMR spectroscopy resulted in a dramatic accumulation of P-choline in the cytoplasmic compartment containing choline kinase and not in the vacuole. The total accumulation of P-choline over a 10-h period exhibited Michaelis-Menten kinetics. During this period, in the absence of Pi in the perfusion medium there was a marked depletion of glucose-6-P, and the cytoplasmic Pi resonance disappeared almost completely. When a threshold of cytoplasmic Pi was attained, the phosphorylation of choline was sustained by the continuous release of Pi from the vacuole although at a much lower rate. However, when 100 microM inorganic phosphate was present in the perfusion medium, externally added Pi was preferentially used to sustain P-choline synthesis. It is clear, therefore, that cytosolic choline kinase associated with a carrier-mediated transport system for choline uptake appeared as effective systems for continuously trapping cytoplasmic Pi including vacuolar Pi entering the cytoplasm.     

Water Transport across Yeast Vacuolar and Plasma Membrane-Targeted Secretory Vesicles Occurs by Passive Diffusion

To determine whether solute transport across yeast membranes was facilitated, we measured the water and solute permeations of vacuole-derived and late secretory vesicles in Saccharomyces cerevisiae; all permeations were consistent with passive diffusive flow. We also overexpressed Fps1p, the putative glycerol facilitator in S. cerevisiae, in secretory vesicles but observed no effect on water, glycerol, formamide, or urea permeations. However, spheroplasts prepared from the strain overexpressing Fps1p showed enhanced glycerol uptake, suggesting that Fps1p becomes active only upon insertion in the plasma membrane.     

Serine-specific phosphorylation of nicotinic receptor associated 43K protein

In Torpedo marmorata electroplaque, an extrinsic membrane protein of apparent mass 43,000 daltons colocalizes with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR) in approximately 1:1 stoichiometry. We show that this 43K protein can be phosphorylated in vitro by endogenous protein kinases present in AChR-rich membranes. The extent of 43K protein phosphorylation exceeds that of the subunits of the AChR, well-established substrates for enzymatic phosphorylation. We demonstrate that significant 43K phosphoprotein exists in vivo. The kinetics of phosphate incorporation mediated by endogenous kinases differed significantly from those of the AChR subunits, suggesting that different phosphorylation cascades are involved. Use of specific inhibitors of a variety of protein kinases indicated that endogenous cAMP-dependent protein kinase catalyzes phosphorylation of the 43K protein in vitro. All of the phosphate incorporated into 43K protein was accounted for by phosphoserine (0.65 mol/mol of 43K protein). Potential structural and functional consequences of 43K protein phosphorylation are discussed.     

Topography of protein kinases and phosphoproteins in the plasma membrane of 3T3 cells

The plasma membrane of 3T3 cells contains at least two different endogenous cyclic AMP-dependent protein kinase systems. One catalyzes the phosphorylation of endogenous protein substrates, i.e., PP24 and PP14, whereas the other catalyzes the phosphorylation of exogenous substrates. In this paper the topography of these cyclic AMP-dependent phosphorylation systems is described. The results show that the kinases which phosphorylate only exogenous substrates are primarily localized to the outer plasma membrane surface whereas the endogenous cyclic AMP-dependent protein kinase and its two endogenous substrates are localized to the cytoplasmic plasma membrane surface. The data also establish that neither the cytoplasmically orientated kinase nor its substrates has a transmembrane orientation even though factors acting on the outer plasma membrane can affect these proteins. This suggests that functional modulation of the cytoplasmically localized cyclic AMP-dependent phosphorylation system can be mediated by a transmembrane regulatory mechanism. The importance of determining the topography of such plasma membrane phosphorylation systems is emphasized by recent studies which show that neoplastic transformation can be mediated at least in part by protein kinases and/or phosphoproteins which are localized on the cytoplasmic surface of the plasma membrane.     

Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli.

The specificity of the glycerol facilitator (glpF) of Escherichia coli was studied with an osmotic method. This transport system allowed the entry of polyols (glycerol and erythritol), pentitols, and hexitols. The analogous sugars were not transported. However, urea, glycine, and DL-glyceraldehyde could use this pathway to enter the cell. The glpF protein allowed the rapid efflux of preequilibrated xylitol. Glycerol surprisingly did not inhibit the uptake of xylitol, and xylitol only slightly reduced the uptake of glycerol. The observation and the insensitivity of the xylitol transport to low temperature suggest that the facilitator behaves as a membrane channel.     

Importance of Facilitated Diffusion for Effective Utilization of Glycerol by Escherichia coli

Wild-type Escherichia coli possesses an inducible permeation system which catalyzes facilitated diffusion of glycerol into the cell. A spectrophotometric method can be used to assess the presence of this mechanism. The structural gene for the facilitator (glpF) and the structural gene for glycerol kinase (glpK) apparently belong to a single operon. The glpF(+) allele permits effective glycerol utilization by the cells, and, at millimolar concentrations of glycerol, cells carrying the glpF(+) allele grow much faster than glpF genotypes. Although the glycerol-scavenging power of the cell depends both on the facilitated entry of the substrate and its subsequent trapping by an adenosine triphosphate-dependent phosphorylation, the two gene products, the facilitator and kinase, function independently. Wild-type Shigella flexneri appears to be glpK(+) but glpF. This organism grows slowly in media at low concentrations of glycerol. When the glpF(+) and glpK(+) alleles of E. coli are inserted into the S. flexneri genome by transduction, the hybrid strain grows rapidly in low glycerol medium. Vice versa, when the glpF and glpK(+) alleles of S. flexneri are incorporated into E. coli, the hybrid strain grows slowly in low glycerol medium.     

Maltose-binding protein does not modulate the activity of maltoporin as a general porin in Escherichia coli.

Maltoporin (lambda receptor) is part of the maltose transport system in Escherichia coli and is necessary for the facilitated diffusion of maltose and maltodextrins across the outer membrane. Maltoporin also allows the diffusion of nonmaltodextrin substrates, albeit with less efficiency. The preference of maltoporin for maltodextrins in vivo is thought to be the result of an interaction of maltoporin with the maltose-binding protein, the malE gene product. In a recent report Heuzenroeder and Reeves (J. Bacteriol. 144:431-435, 1980) suggested that this interaction establishes a gating mechanism which inhibits the diffusion of nonmaltodextrin substrates, such as lactose. To reinvestigate this important conclusion, we constructed ompR malTc strains carrying either the malE+ gene, the nonpolar malE444 deletion, or the malE254 allele, which specifies an interaction-deficient maltose-binding protein. Lactose uptake was measured at different concentrations below the Km of this transport system and under conditions where transport was limited by the diffusion through maltoporin. We found no difference in the kinetics of lactose uptake irrespective of the malE allele. We conclude that the maltose-binding protein does not modulate the activity of maltoporin as a general outer membrane porin.     

Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.     

cAMP-dependent protein kinase A selects the excited state of the membrane substrate phospholamban

Phosphorylation of membrane proteins is a central regulatory and signaling mechanism across cell compartments. However, the recognition process and phosphorylation mechanism of membrane-bound substrates by kinases are virtually unknown. cAMP-dependent protein kinase A (PKA) is a ubiquitous enzyme that phosphorylates several soluble and membrane-bound substrates. In cardiomyocytes, PKA targets phospholamban (PLN), a membrane protein that inhibits the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA). In the unphosphorylated state, PLN binds SERCA, reducing the calcium uptake and generating muscle contraction. PKA phosphorylation of PLN at S16 in the cytoplasmic helix relieves SERCA inhibition, initiating muscle relaxation. Using steady-state kinetic assays, NMR spectroscopy, and molecular modeling, we show that PKA recognizes and phosphorylates the excited, membrane-detached R-state of PLN. By promoting PLN from a ground state to an excited state, we obtained a linear relationship between rate of phosphorylation and population of the excited state of PLN. The conformational equilibrium of PLN is crucial to regulate the extent of PLN phosphorylation and SERCA inhibition.     

Intramolecular and intermolecular enzymatic modulation of ion channels in excised membrane patches.

A calcium-activated potassium channel in posterior pituitary nerve terminals was modulated by phosphorylation and dephosphorylation. Nearly every patch of membrane containing this channel also contained both membrane bound protein phosphatase and membrane-bound protein kinase. By examining the statistical and kinetic nature of phosphorylation and dephosphorylation in excised patches, it was possible to evaluate two contrasting models for these enzymatic reactions. One of these models treated catalysis as an intermolecular process in which the enzyme and substrate are separate molecular species that diffuse and encounter one another during collisions. The second model treated catalysis as an intramolecular process in which the enzyme and substrate reside within a stable macromolecular complex. The study began with a Poisson analysis of the distribution of channel number in patches, and of the number of protein phosphatase-free and protein kinase-free patches. Subsequent kinetic analysis of dephosphorylation yielded an estimate of the mean number of protein phosphatase molecules per patch that was similar to the value obtained from Poisson analysis. Because these two estimates were independent predictions based on the intermolecular model, their agreement supported this model. Analysis of channel number in protein phosphatase-free patches and of the rarity of patches showing partial but incomplete rundown provided additional support for the intermolecular model over the intramolecular model. Furthermore, dephosphorylation exhibited monotonic kinetics with a rate well below the diffusion limit. Thus, several different lines of analysis support the intermolecular model for dephosphorylation, in which the protein phosphatase must encounter its substrate to effect catalysis. In contrast to the monotonic kinetics of dephosphorylation, the phosphorylation reaction exhibited sigmoidal kinetics, with a rate that depended on membrane potential. Voltage dependence is an unlikely property for a kinetic step involving encounters resulting from diffusion. Furthermore, the velocity of the phosphorylation reaction exceeded the diffusion limit, and this observation is inconsistent with the intermolecular model. Thus, both intermolecular and intramolecular enzymatic mechanisms operate in the modulation of the calcium-activated potassium channel of the posterior pituitary. These studies provide a functional characterization of the interactions between enzyme and substrate in intact patches of cell membrane.