Evolution is a key framework upon which scientists interpret biological data. The genomics revolution is so rapidly expanding that we utterly depend on the recently founded disciplines of bioinformatics and biosystematics to interpret the available information. In particular, we use these disciplines to better understand the evolutionary histories of transport proteins.

Transmembrane molecular transport is essential to every living cell. The proteins that catalyze transport (www.tcdb.org) allow the entry of all essential nutrients into the cell and its compartments, regulate concentrations of metabolites by both uptake and excretion mechanisms, provide ion concentration gradients that generate electrical potentials, export macromolecules, catalyze export and uptake of signaling molecules that mediate intercellular and intracellular communication, prevent toxic effects of poisons by catalyzing their active efflux, and export biologically active agents that damage or kill other cells. Thus, transport underlies all life-endowing processes, including metabolism, communication, biosynthesis, reproduction, and both cooperative and antagonistic behaviors.

To establish that different membrane proteins are polyphyletic, i.e., that they arose independently of each other, we need to establish distinct routes of their evolutionary appearances. Several developments make this effort feasible, including sensitive software, the chance to analyze large numbers of homologues from genome sequencing, and application of the Superfamily Principle. This principle, established by Russell Doolittle at the University of California in San Diego in 1981, holds that if macromolecule A is homologous to macromolecule B, meaning that they both derived from a common ancestor, and B is homologous to C, then A is also homologous to C. Despite the simplicity of this precept, some molecular biologists still question its validity.

To establish homology between two or more proteins, one can invoke the Superfamily Principle along with statistical approaches, but to establish a lack of homology, one must show that the two proteins evolved independently. For instance, the ABC and PTS functional superfamilies were recently shown to consist of different families of porters that lack homology between families. At least three distinct families of transport proteins occur in each of these superfamilies.

The ABC Superfamily

The ATP-binding cassette (ABC) superfamily is considered one of the two largest superfamilies of transmembrane transporters. The ABC exporters (Fig. 1A) and importers (Fig. 1B) have distinctive generalized structures. By tradition, the ABC superfamily is defined on the basis of its energy-coupling proteins, the monophyletic ATP-hydrolyzing proteins or protein domains, which all share a single common evolutionary origin (green subunits [C], Fig.1). Phylogenetic analyses indicate that these subunits segregate according to polarity (direction) of transport, and that the C subunits that function with exporters segregate according to the topological type of membrane subunits (ABC1, 2 or 3) that they energize.

The integral membrane porters or porter domains (red subunits [M], Fig. 1) provide the basis for classifying most other transport protein families. In some cases, such as ABC uptake transporters, additional components, the extracytoplasmic receptors (R, blue, Fig. 1B), are essential for efficient transport activity. The three constituents—M, C, and R—can, but need not be, fused together, giving rise to multidomain transporters.

We have wondered whether the membrane constituents of ABC transporters derived from a single ancestral protein. If they are all homologous, a single transport mechanism and common structural features can be predicted. However, if ABC porters are polyphyletic, there is no basis for extrapolating findings made with one phylogenetic group of proteins to another.

Independent Origins for ABC Porters

We know of 86 families of ABC transporters, 33 for solute uptake and 53 for solute export (www.tcdb.org). They can transport small molecules such as nutrients, salts, and toxins, or they can function in macromolecular efflux, secreting proteins, complex carbohydrates, and lipids. The integral membrane components (red subunits, Fig. 1) belong to three topological or structural types called ABC1, ABC2, and ABC3. All three types (Fig. 2) are present in all three domains of living organisms, although one of them (ABC3) has been found only in lower, but not in higher eukaryotes.

The three repeats of two transmembrane α-helical segments (TMSs), which are found in the 6 TMS ABC1 porters, arose from a primordial 2-TMS hairpin-encoding genetic element by intragenic triplication. That process yielded a protein consisting of three tandem transmembrane hairpins, all in the same polypeptide chain and all with the same orientation in the membrane (Fig. 2A). By contrast, the ABC2 porters arose from a primordial 3 TMS-encoding genetic element by intragenic duplication, yielding 6 TMS proteins with two homologous halves that are oppositely oriented in membranes (Fig. 2B). Finally, ABC3 porters can have 4, 8, or 10 putative TMSs. The 4 TMS-encoding genetic element, present in tandem as a pair in some ABC3 transporters, intragenetically duplicated to yield the 8 and 10 TMS proteins, always with the two homologous 4 TMS segments oriented in the same way in the membrane (Fig. 2C). Surprisingly, in the 10 TMS proteins, the two repeated units are separated by two extra, nonhomologous TMSs that apparently arose during or after that duplication.

While the ABC1 exporter type is found in larger numbers than the ABC2 exporter type, all ABC uptake systems appear to be of the ABC2 type. We postulate that the ABC3 porters arose relatively late, as the repeat units in the 8 and 10 TMS proteins have a high degree of sequence identity compared to the smaller repeat units in the ABC1 and ABC2 types. Moreover, the distribution of ABC3 proteins is much more restricted than the other two topological types.

High-resolution X-ray structures for several ABC importers show them all to have a similar three-dimensional fold. However, this structure differs from that of the exporters, which share similar three-dimensional structures. This observation is in agreement with our conclusion of multiple origins for ABC1-3 porters because the importers are of the ABC2 type, while all the analyzed exporters are of the ABC1 type. Structures of ABC2 and ABC3 exporters are not yet available. X-ray crystallographers therefore have their work cut out for them.We expect their analyses to confirm our bioinformatic conclusions about these porters.

The Phosphoenolpyruvate-Dependent Sugar Transporting System

Transporter components of the phosphoenolpyruvate- dependent sugar-transporting phosphotransferase system (PTS) are structurally even more complex than are the ABC transporters (Fig. 3). This fact reflects the complexity of the system, which has multiple functions. For example, it catalyzes sugar transport and phosphorylation and regulates both gene expression and metabolism by several mechanisms. As a protein kinase system, it regulates other cellular activities by phosphorylating a variety of proteins.

A PTS permease complex generally consists of five proteins, called Enzyme I, HPr, IIA, IIB, and IIC (Fig. 3). The ultimate phosphoryl donor is phosphoenolpyruvate (PEP), which phosphorylates Enzyme I, an enzyme with extensive sequence similarity to an enzyme of gluconeogenesis, PEP synthase. The phosphoryl group of Enzyme I is transferred to HPr, and then sequentially to two constituents of the Enzyme II complex, IIA and IIB. Only when IIB is phosphorylated can the sugar be transported into the cell and simultaneously phosphorylated at the expense of IIB-phosphate. Surprisingly, the Enzymes IIA and IIB do not all have the same fold, according to X-ray crystallographic data. Instead, there are three structurally dissimilar IIAs and three structurally dissimilar IIBs. Therefore, the IIA and IIB constituents appear to be polyphyletic (see table).

Independent Origins for PTS Permeases

Recent evidence suggests that the IIC components of PTS permeases are also polyphyletic. There are three evolutionarily distinct families of PTS porters. Members of the first, called the glucose (Glc)-fructose (Fru)-lactose (Lac) family, transport a wide range of sugars and have a uniform topology of eight TMSs per polypeptide chain. These porters, which function with two of the three recognized types of IIA and IIB proteins, were probably the first to evolve. The fructose systems are thought to be primordial because many bacteria have only this PTS permease. Additionally, only fructose-phosphate feeds directly into glycolysis without modification, and the PTS may have evolved as a component of glycolysis.

The ascorbate (Asc)- galactitol (Gat) family consists of members with 12 TMSs showing no sequence similarity to members of the Glc-Fru-Lac family. Their properties (see table) suggest that the Gat porters can function either as secondary carriers or as PTS-coupled systems, while Asc porters can function only when PTS-coupled. These 12 TMS IIC permeases function with IIA and IIB constituents derived from those of the Glc-Fru-Lac family, and they apparently evolved by duplication of a 6 TMS precursor.

Finally, the membrane IIC constituents of the third family, the mannose (Man) family, consist of six TMS proteins showing no significant sequence similarity to proteins of the other two families. In fact, their IIA and IIB constituents are also unrelated to those of the other two families. It seems that all constituents of the mannose PTS transporters evolved independently of those of the other two families of PTS porters.

Perspective

Recent bioinformatic analyses reveal that two of the largest functional superfamilies of transporters, ABC-type primary active transporters and PTS-type substrate phosphorylating transporters, are true mosaic systems. Both consist of at least three families of permease subunits or domains that evolved independently of each other. Their sole common feature is their use of the same energy-coupling proteins, the ATP hydrolyzing subunits of ABC transporters and the common phosphoryl transfer proteins, Enzyme I and HPr, of PTS porters.

New evidence is revealing that in both superfamilies the membrane porters alone may catalyze transport without energy coupling involving ATP or PEP. Thus, the primordial transport proteins upon which a chemical form of energy was superimposed may still exist. Further studies will be required to determine to what degree these observations are applicable to other transporters. Nevertheless, the bioinformatic analyses provide guides for future structural, mechanistic, and physiological investigations.


SUGGESTED READING

Doolittle, R. F. 1981. Similar amino acid sequences: chance or common ancestry? Science 214:149-159.

Khwaja, M., Q. Ma, and M.H. Saier, Jr. 2005. Topological analysis of integral membrane constituents of prokaryotic ABC efflux systems. Res. Microbiol. 156:270-277.

Peterkofsky, A., G. Wang, D. S. Garrett, B. R. Lee, Y. J. Seok, and G. M. Clore. 2001. Three-dimensional structures of protein-protein complexes in the E. coli PTS. J. Mol. Microbiol. Biotechnol. 3:347-354.

Rodionov, D. A., P. Hebbeln, A. Eudes, J. ter Beek, I. A. Rodionova, G. B. Erkens, D. J. Slotboom, M. S. Gelfand, A. L. Osterman, A. D. Hanson, and T. Eitinger. 2009. A novel class of modular transporters for vitamins in prokaryotes. J. Bacteriol. 191:42-51.

Saier, M. H., Jr., R. N. Hvorup, and R. D. Barabote. 2005. Evolution of the bacterial phosphotransferase system: from carriers and enzymes to group translocators. Biochem. Soc. Trans. 33:220-224.

Saier, M. H., Jr., M. R. Yen, K. Noto, D. G. Tamang, and C. Elkan. 2009. The Transporter Classification Database: recent advances. Nucleic Acids Res. 37:D274-278.

Wang, B., M. Dukarevich, E. I. Sun, M. R. Yen, and M. H. Saier, Jr. 2009. Membrane porters of ATP-binding cassette (ABC) transport systems are polyphyletic. J. Membr. Biol. (PMID 19806386).

Yen, M. R., J. Choi, and M. H. Saier, Jr. 2009. Bioinformatic analyses of transmembrane transport: novel software for deducing protein phylogeny, topology, and evolution. J. Mol. Microbiol. Biotechnol. 17:163-176.

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