Introduction

The galactose permease or GalP found in Escherichia coli

Escherichia coli

Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

is an integral membrane protein

Integral membrane protein

An integral membrane protein is a protein molecule that is permanently attached to the biological membrane. Proteins that cross the membrane are surrounded by "annular" lipids, which are defined as lipids that are in direct contact with a membrane protein...

 involved in the transport of monosaccharides, primarily hexose

Hexose

In organic chemistry, a hexose is a monosaccharide with six carbon atoms, having the chemical formula C6H12O6. Hexoses are classified by functional group, with aldohexoses having an aldehyde at position 1, and ketohexoses having a ketone at position 2....

s, for utilization by E. coli in glycolysis

Glycolysis

Glycolysis is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+...

 and other metabolic and catabolic pathways (3,4). It is a member of the Major Facilitator Super Family (MFS) and is homologue of the human GLUT1

GLUT1

Glucose transporter 1 , also known as solute carrier family 2, facilitated glucose transporter member 1 is a protein that in humans is encoded by the SLC2A1 gene...

 transporter (4). Below you will find descriptions of the structure, specificity, effects on homeostasis, expression, and regulation of GalP along with examples of several of its homologues.

Structure

Galactose Permease (GalP), is a member of the Major Facilitator Super Family (MFS) and therefore has structural similarities to the other members of this super family such as GLUT1

GLUT1

Glucose transporter 1 , also known as solute carrier family 2, facilitated glucose transporter member 1 is a protein that in humans is encoded by the SLC2A1 gene...

 (4). All members of the MFS have 12 membrane spanning alpha(α)-helices with both the C- and N-termini located on the cytoplasmic side of the membrane (4). Figure 1a (3) depicts how the 12 helices are divided into two halves, that are pseudo-symmetric, of 6 helices which are attached by a long hydrophilic cytoplasmic loop between helix 6 and helix 7 (2,3,4). These two halves come together to form a pore for substrate transport, in GalP, the substrates are primarily galactose

Galactose

Galactose , sometimes abbreviated Gal, is a type of sugar that is less sweet than glucose. It is a C-4 epimer of glucose....

, glucose, and H+. GalP monomers have a pore of approximately 10Å in diameter, which is consistent with the pore sizes found in other members of the MFS, between 10-15Å (4). GalP has been found as an oligomer formed by a homotrimer of GalP monomers that exhibits p3 or 3-fold rotational symmetry (Figure 1b-c) (4). GalP is the first member of the MFS that has been found as a trimer and to be biologically active in its trimeric form; it is thought that the GalP oligomer is formed for stability (4).

Specificity

GalP is a monosaccharide transporter that uses a chemiosmotic

Chemiosmosis

Chemiosmosis is the movement of ions across a selectively permeable membrane, down their electrochemical gradient. More specifically, it relates to the generation of ATP by the movement of hydrogen ions across a membrane during cellular respiration....

 mechanism to transport its substrates into the cytoplasm of E. coli (1). Glucose, galactose and other hexoses are transported by GalP by the use of the proton gradient produced by the electron transport chain and reversible ATPase

ATPase

ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...

 (1). GalP can bind specifically to the hexoses with preferential binding of galactose and glucose through the pores in each monomer (2,3). It transports these sugars at faster rates with a proton gradient but can still transport them in a leaky fashion without a proton gradient present (4). As stated before GalP shares similarities with GLUT1

GLUT1

Glucose transporter 1 , also known as solute carrier family 2, facilitated glucose transporter member 1 is a protein that in humans is encoded by the SLC2A1 gene...

 and other members of the MFS and like GLUT1, GalP can be inhibited by the antibiotics cytochalasin B

Cytochalasin B

Cytochalasin B is a cell-permeable mycotoxin. It inhibits cytoplasmic division by blocking the formation of contractile microfilaments. It inhibits cell movement and induces nuclear extrusion. Cytochalasin B shortens actin filaments by blocking monomer addition at the fast-growing end of polymers....

 and forskolin

Forskolin

Forskolin is a labdane diterpene that is produced by the Indian Coleus plant . Forskolin is commonly used to raise levels of cyclic AMP in the study and research of cell physiology. Forskolin resensitizes cell receptors by activating the enzyme adenylyl cyclase and increasing the intracellular...

 (Figure 1a) (3), which competitively bind to the pore blocking sugar transport into the cell (2,3,4). Forskolin is a structural homologue of D-galactose (Figure 1a) (3) and therefore can bind with a similar affinity to the transporter. Cytochalasin B may bind to an asparagine residue (Asn394) in the pore, blocking saccharide uptake, which is also found in the GLUT1 transporter (2,3). GalP can transport lactose or fructose but with low affinity, only allowing these sugars to "leak" across the membrane when glucose, galactose, or other hexoses aren't present for transport (4).

Homeostasis

The GalP symporter

Symporter

A cotransporter is an integral membrane protein that is involved in secondary active transport. It works by binding to two molecules or ions at a time and using the gradient of one solute's concentration to force the other molecule or ion against its gradient....

 links galactose and proton import, using the favorable proton concentration gradient to move galactose against its concentration gradient. However, this mechanism, if in isolation, would result in acidification of the cytoplasm and cessation of galactose import(14). To prevent this, E. coli utilizes ion pumps designed to raise intracellular pH (13,14). During electron transport (a key step in ATP production in respiration

Cellular respiration

Cellular respiration is the set of the metabolic reactions and processes that take place in the cells of organisms to convert biochemical energy from nutrients into adenosine triphosphate , and then release waste products. The reactions involved in respiration are catabolic reactions that involve...

), energy harnessed from electrons is used to pump protons into the periplasmic space to build a proton motive force. Primary proton pumps, responsible for pumping protons out of the cytoplasm, can be active without the synthesis of ATP

Adenosine triphosphate

Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...

 and are the primary mechanism through which protons are exported (13,14). Coupling galactose/proton import with proton export would maintain pH homeostasis

Homeostasis

Homeostasis is the property of a system that regulates its internal environment and tends to maintain a stable, constant condition of properties like temperature or pH...

. As protons are charged molecules, their import or export could disrupt the membrane potential of the cell (14). However, simultaneous import and export of protons would result in no change in the net charge of the cell, thus no net change in membrane potential.

Regulation/Expression

The GalP/H+ symporter is the galactose permease from the galP gene of the Eschericha coli genome. Galactose is an alternate carbon source to the preferable glucose . The cAMP/CRP catabolite repression regulator is most likely involved in the regulation of GalP expression (Figure 2) (9). The two proteins responsible for inhibiting transcription from the gal regulon are GalR and GalS (Figure 4) (11). GalR and GalS have very similar primary structure sequences, and have the same binding sites on the operator (11). In the presence of D-galactose, GalR and GalS are inhibited since they are repressors (5, 11). However, when GalP is not required (i.e. when glucose is available), GalR/GalS will bind the promoter operator site thus blocking transcription and preventing cAMP-CRP activation (11). GalS is seen to bind only in the presence of GalR, so both of these proteins are required for repression (11). cAMP is what modulates CRP at the promoter. The cAMP-CRP complex activates the gal regulon and is responsible for upregulation of GalP (Figure 2) (9,11). GalP is also repressed in the presence of glucose since the cell will prefer glucose over galactose (7).
There is also a study on the involvement of NagC in regulation, a protein from the nagC gene which is responsible for N-acetylglucosamine repression (5). This study suspects that NagC cooperates with GalR and GalS by binding to a single-high affinity site upstream of the galP promoter as well in order to suppress gal regulon transcription (5).

Other Bacteria Symporters

Several other symporter

Symporter

A cotransporter is an integral membrane protein that is involved in secondary active transport. It works by binding to two molecules or ions at a time and using the gradient of one solute's concentration to force the other molecule or ion against its gradient....

s have been identified in E. coli and in other bacteria. E. coli has a well-studied GltS glutamate/Na+ symporter that aids in the uptake of glutamate into the cell along with an influx of sodium ions. It also has a serine-threonine symporter, SstT, that also uses an influx of sodium ions for solute uptake.

A Na+/glucose symporter (SglT) has been identified in Vibrio parahaemolyticus (10). Sodium ions induced the cells’ uptake of glucose in a study of phosphotransferase-system (PTS)

PEP group translocation

PEP group translocation, also known as the phosphotransferase system or PTS, is a distinct method used by bacteria for sugar uptake where the source of energy is from phosphoenolpyruvate . It is known as multicomponent system that always involves enzymes of the plasma membrane and those in the...

 mutants (10). Clostridum difficle has a symporter homologous to that of the V. parahaemolyticus SglT (6). A citrate/Na+ symporter, CitS, seems to be common between Vibrio cholerae, Salmonella Typhi, and Klebsiella pneumoniae (6). This symporter uses the influx of sodium ions in order to bring citrate into the cell, which is an important substrate to have for metabolic processes such as decarboxylation of oxaloacetate

Citric acid cycle

The citric acid cycle — also known as the tricarboxylic acid cycle , the Krebs cycle, or the Szent-Györgyi-Krebs cycle — is a series of chemical reactions which is used by all aerobic living organisms to generate energy through the oxidization of acetate derived from carbohydrates, fats and...

 (6). A H+/amino acid symporter BrnQ can be found in Lactobacillus delbruckii, and Pseudomonas aeruginosa has the BraB symporter for substrates such as glutamate as well (6).

Solute/ion symporters are very commonly found in bacteria since they are very important. Homeostasis

Homeostasis

Homeostasis is the property of a system that regulates its internal environment and tends to maintain a stable, constant condition of properties like temperature or pH...

 and regulated uptake for metabolic pathways is essential for bacterial survival.

GLUT-1: A Eukaryotic Homolog

GalP is homologous to GLUT-1 found in mammalian cells (12). Both transporters are MFS transporters and possess 29% sequence identity (4). GLUT-1 is a glucose transporter present in most mammalian cells (Figure 5) (12). Its structure is nearly identical to that of GalP – possessing cytoplasmic amino and carboxy termini, twelve membrane spanning α helices, a periplasmic glycosylation site between helices 1 and 2, and a cytoplasmic α-helix loop between helices 6 and 7 (12). GLUT-1 ranges from 45 to 55 kDa; the size variation depends upon the extent of glycosylation

Glycosylation

Glycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule . In biology glycosylation refers to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules...

 (12).

While GLUT-1 is found in most mammalian cells, certain tissue types express this transporter more so than others. GLUT-1 is expressed in high levels on erythrocytes, embryonic cells, fibroblasts, and endothelial cells (12). GLUT-1 is also one of the main transporters involved in transporting glucose across the blood brain barrier (12).

Generally, GLUT-1 acts as a facilitative transporter of glucose, transporter glucose along its concentration gradient. When glucose binds to GLUT-1, it stimulates a conformational change

Conformational change

A macromolecule is usually flexible and dynamic. It can change its shape in response to changes in its environment or other factors; each possible shape is called a conformation, and a transition between them is called a conformational change...

, allowing glucose to be released on the opposite side of the membrane (4,12). GLUT-1 is a bidirectional transporter and possesses glucose binding sites accessible on both the cytoplasmic and extracellular faces (4,12). On the rare occasion that GLUT-1 transports glucose against its concentration gradient, Glut-1 uses an energy source, typically ATP

Adenosine triphosphate

Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...

, to move the glucose. Like GalP, GLUT-1 is inhibited via the binding of cytochalasin B

Cytochalasin B

Cytochalasin B is a cell-permeable mycotoxin. It inhibits cytoplasmic division by blocking the formation of contractile microfilaments. It inhibits cell movement and induces nuclear extrusion. Cytochalasin B shortens actin filaments by blocking monomer addition at the fast-growing end of polymers....

 and forskolin

Forskolin

Forskolin is a labdane diterpene that is produced by the Indian Coleus plant . Forskolin is commonly used to raise levels of cyclic AMP in the study and research of cell physiology. Forskolin resensitizes cell receptors by activating the enzyme adenylyl cyclase and increasing the intracellular...

(12).