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APC Superfamily

The amino acid/polyamine/organocation (APC) superfamily is among the largest transport superfamilies identified. It is found in all forms of life. However, not much is known about the structures of members of this family, because not many proteins have been crystallized. These few structures lend much insight into the qualities of the superfamily as a whole when compared with existing hydropathy plots and other structural studies.

Superfamilies have been identified using phylogenetic analysis. Thus, only proteins with similar genetic qualities and evolutionary roots are considered members of the same superfamily. Because of this, proteins with similar topographies are not necessarily in the same superfamily. For instance, the core structures of ApcT and AdiC, members of the APC superfamily discussed below, are very similar to that of LeuT (a member of the neurotransmitter sodium symporter (NSS) family), BetP (a member of the betaine/chlorine/carnitine transporter (BCCT) family), vSGLT (of the solute sodium symporter (SSS) family) and Mhb1 (of the nucleobase-cation-symport-1 (NCS1) family)[1]

Various structural studies have been conducted outside of crystallography. According to hydropathy plots [2], all members of the APC superfamily exhibit a uniform topology formed by a single polypeptide chain that crosses the plasma membrane 12 times, unless otherwise noted. All of these proteins also share a similar structural core, consisting of two V-shaped domains of five transmembrane domains each, intertwined in an antiparallel topology. [3] Each protein's N- and C-termini are located in the cytosol. The loops in the cytosol tend to be smaller than the loops located in the extracellular space. The smaller proteins are generally of prokaryotic origin, whereas the larger ones are of eukaryotic origin and have N- and C-terminal hydrophilic extensions.

Functional studies have revealed that some proteins in this superfamily exhibit broad specificity while others respond to only one or a few substrates.

ApcT - a proton-coupled amino acid transporter

ApcT has not been placed in a particular family within the APC superfamily. It is a proton-coupled amino acid transporter.

ApcT in high pH, inward facing, apo conformation [3GIA]

Drag the structure with the mouse to rotate
where the N-terminus is gradually shaded into the C-terminus according to the scale below.
N               C

ApcT is cylindrical in shape with 12 TM domains (numbered from N-terminus to C-terminus), short intracellular and extracellular loops, and each termini ending in the cytoplasm, consistent with the topography of AdiC described below.

ApcT is a proton-coupled, broad specificity transporter. It transports a range of amino acids (Glu, Ala, > Ser, Gln > Phe,) but transports the smallest (Gly) or largest (Trp) amino acids very slowly, if at all.

Lys-158, has been proposed to be necessary for proton coupled transport in this protein. Because the displayed structure was collected in alkaline conditions, it is assumed that this is the conformation of the protein when Lys-158 is not protonated. Upon protonation of the Lys-158 amine group, it is expected that ApcT will isomerize from this inward facing state to an outward facing conformation, ready to bind the substrate and return to an inward facing conformation to release/exchange it. Experiments with ApcT established that the occluded formation, a formation in which the substrate binding pocket is protected from the protein's environment, is not dependent on substrate binding [4].

Ten Established Protein Families

AAT: Amino Acid Transporter

Unique to bacteria, this is the largest family within the APC superfamily. Members of this family have short hydrophilic extensions at both termini. One example of a protein in this family is GabP, a γ-Aminobutyrate permease isolated from both Bacillus subtilis and Escherichia coli . GabP appears to follow the APC topography of 12TMs and cytoplasmic termini, based on hydropathy and experimental analysis [5].

APA: Basic Amino Acid/Polyamine Transporter

The APA family is also unique to bacteria. For example, ArcD, an arginine:orithine transporter has been identified in Chlamydophilia pneumoniae and Chlamydia trachomatis .

AdiC - an example from the APA family

AdiC has been determined to be a member of the APC superfamily through phylogenetic analysis. [6]

AdiC is a representative member of the APC superfamily; it contains 12 TMs with each termini located in the cytosol. AdiC functions as an arginine/agmatine antiporter in E. coli, Salmonella enterica serovar Typhimurium[7], and other bacteria when the cell is exposed to acidic environments. Agmatine is the product of arginine decarboxylation. An associated protein, AdiA[8], is thought to decarboxylate arginine molecules for transport. By exchanging intracellular agmatine (Agm(2+)) for extracellular arginine (Arg(+)), AdiC removes virtual protons from the interior of the cell, enabling the bacteria to survive in acidic conditions by preventing acidification of the cytosol.

Outward-facing AdiC homodimer [3LRB]

Drag the structure with the mouse to rotate
Though the structure is shown as a homodimer, each subunit is an independently functioning transporter. [9]

As in ApcT, 10 of the transmembrane helices surround the pore in antiparallel fashion, with TM1 paired with TM6, TM2 with TM7, and so on. TMs 11 and 12 do not participate in this pairing scheme; instead, they form most of the homodimeric interface. TMs 1 and 6 contain short, non-helical, Gly-containing loops midway across the membrane, thought to be important for substrate interactions [10]. Amino acids in these loops are highly conserved within distinct families of the APC superfamily. For example, AdiC, CadB, and PotE, which all belong to the APA family, share two signature loop sequences, the GSG motif (Gly25-Ser26-Gly27) in TM1 and the GVESA motif (Gly206-Val-Glu-Ser-Ala210) in TM6.

AdiC in complex with Arginine and B-Nonylglucoside [3L1L]

Drag the structure with the mouse to rotate
Arg binding induces a structural rearrangement in TM6 and minor changes in TM2 and TM10, resulting in an occluded formation [11].

Trp-293 has been demonstrated to be vital for substrate recognition [12]. Trp-293 is buried in the substrate-binding site, Notice the aromatic interactions with the substrate. Tyr-93 is another residue proposed to be vital for substrate specificty and/or binding, Trp-293 and Tyr-93 come from TM8 and TM3, respectively.
Glu-208 also appears to have a role in regulating the upload and release of substrate by acting as a pH sensor. In the stomach (pH 2-3), Glu-208 (pKa approx 4.25) is likely protonated while other residues with a pKa of approx 2 are mostly unprotonated. Thus, the majority of these amino acids carry no net charge on their head groups, with the positively charged a-amino group offsetting the negatively charged a-carboxyl group. Protonated Glu-208 likely binds to the neutral head groups of the substrate amino acid (arginine). Once facing the intracellular environment (pH 4-5), Glu-208 is likely to deprotonate, developing a net negative charge in the substrate-binding cavity. The negative charge may attract positively charged head group of the a-decarboxylated amino acid (agmatine) and favor its binding. When this occurs, agmatine replaces arginine in the binding site, releasing arginine into the cytosol and initiating the deportation of the agmatine.

CAT: Cationic Amino Acid Transporter

Members of the CAT family are ubiquitous, containing 14 TMs in eukaryotes and 12 TMs in prokaryotes. These proteins have short, hydrophilic, N-terminal extensions. CAT2 provides arginine for NO synthesis and arginase in classical and alternative activation of macrophages. CAT1 is required for macrophage proliferation [13]. Members of this family have been shown to serve as viral receptors in animals.

ACT: Amino Acid/Choline Transporter

Members of the ACT family can be found in yeast, plants, and fungi. These proteins have short hydrophilic extensions at the C and N termini.

EAT: Ethanolamine Transporter

Members of the EAT family are found in bacteria. They have no noticeable extensions beyond the 12 TMs. Ethanolamine permeases from this family have been identified in both Rhodococcus erythropolis - a bacterium found in crude oil[14], and Zymomonas mobilis.

ABT: Archaeal/Bacterial Transporter

As the name suggests, members of the ABT family are found in archaea and bacteria. One member (Cat1 Afu) of this family exhibits a long, C-terminal extension that may function in interactions with other proteins.

GGA: Glutamate:GABA Antiporter

Members of the GGA family are found only in bacteria. There proteins have short, hydrophilic, N-terminal extensions.

LAT: L-type Amino Acid Transporter

Members of the LAT family have been identified in animals and yeast. The LAT proteins correspond to the light chains in heteromeric amino acid transporters (HATs) in eukaryotes. A HAT is composed of two subunits: one membrane protein (the light subunit) and a disulfide-linked N-glycosylated type II membrane glycoprotein (the heavy subunit). The light subunit is the catalytic component of the transporter, whereas the heavy subunit appears to be vital only for trafficking substrates to the plasma membrane [15].

LAT1 has been found to be overexpressed in a wide variety of primary human cancer and is important to cell growth and survival in cancer cell lines. [16]

SteT - an example from the LAT family

SteT (Serine/Threonine antiporter) from Bacillus subtilis is the first characterized prokaryotic member of the LAT family, put into this family by phylogenetic analysis [17]. Transmission electron microscopy revealed elliptical particles (diameters 6 × 7 nm) with a distinct central depression. Comparing the apparent structure of SteT with that of AdiC and ApcT as well as performing functional analysis through mutagenesis studies suggests the involvement of different TM8 residues in substrate binding and translocation in the LAT transporter SteT:

  • Cys-291 (structurally equivalent to Ser-289 in AdiC) is close to the substrate binding site and possibly directly interacts with the substrate L-Serine.
  • Lys-295 (equivalent to Trp-293 in AdiC) is a determinant of substrate selectivity.
  • S-thiolation of Gly-294 (equivalent to Gly-292 in AdiC) blocks substrate translocation.

The large change in the amino acid substrate specificity of SteT as a result of single mutations in residue Lys-295 demonstrates the plasticity of the substrate binding site of the LAT family (and the APC superfamily). [18] TM1, TM2, and the re-entrant loop between TM2 and TM3 were found to be conserved between the prokaryotic SteT and eukaryotic LAT family members.

SPG: Spore Germination Protein

Members of this family are found in prokaryotes and exhibit only 10 transmembrane segments. The 2 segments closest to the C-terminus in other members of this super family appear to have been cleaved when this family was evolving. None of the proteins in this family have been identified as transporters, leading to the possibility that transmembrane segments 11 and 12 are vital for transport function.

YAT: Yeast Amino Acid Transporter

Members of the YAT family have been identified in both yeast and fungi. Some members of this family exhibit long, N-terminal, hydrophilic extensions beyond the 12 TMs. Examples of this family include lysine-specific permease, proline-specific permease, and tryptophan permease all isolated from Saccharomyces cerevisiae. Another amino acid transporter (Aat1 Amu) isolated from Amanita muscaria also belongs to the YAT family.

References

  1. ↑ Lolkema JS, Slotboom DJ. The major amino acid transporter superfamily has a similar core structure as Na+-galactose and Na+-leucine transporters. Mol Membr Biol. 2008 Sep;25(6-7):567-70. Epub 2008 Nov 21. PMID:19031293 doi:10.1080/09687680802541177
  2. ↑ Jack DL, Paulsen IT, Saier MH. The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations. Microbiology. 2000 Aug;146 ( Pt 8):1797-814. PMID:10931886
  3. ↑ Vangelatos I, Vlachakis D, Sophianopoulou V, Diallinas G. Modelling and mutational evidence identify the substrate binding site and functional elements in APC amino acid transporters. Mol Membr Biol. 2009 Aug;26(5):356-70. Epub 2009 Aug 7. PMID:19670073 doi:913775369
  4. ↑ Shaffer PL, Goehring A, Shankaranarayanan A, Gouaux E. Structure and Mechanism of a Na+ Independent Amino Acid Transporter. Science. 2009 Jul 22. PMID:19608859
  5. ↑ Hu LA, King SC. Membrane topology of the Escherichia coli gamma-aminobutyrate transporter: implications on the topography and mechanism of prokaryotic and eukaryotic transporters from the APC superfamily. Biochem J. 1998 Nov 15;336 ( Pt 1):69-76. PMID:9806886
  6. ↑ Casagrande F, Ratera M, Schenk AD, Chami M, Valencia E, Lopez JM, Torrents D, Engel A, Palacin M, Fotiadis D. Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily. J Biol Chem. 2008 Nov 28;283(48):33240-8. Epub 2008 Sep 25. PMID:18819925 doi:10.1074/jbc.M806917200
  7. ↑ Smith CB, Graham DE. Outer and inner membrane proteins compose an arginine-agmatine exchange system in Chlamydophila pneumoniae. J Bacteriol. 2008 Nov;190(22):7431-40. Epub 2008 Sep 12. PMID:18790867 doi:10.1128/JB.00652-08
  8. ↑ Kieboom J, Abee T. Arginine-dependent acid resistance in Salmonella enterica serovar Typhimurium. J Bacteriol. 2006 Aug;188(15):5650-3. PMID:16855258 doi:10.1128/JB.00323-06
  9. ↑ Fang Y, Jayaram H, Shane T, Kolmakova-Partensky L, Wu F, Williams C, Xiong Y, Miller C. Structure of a prokaryotic virtual proton pump at 3.2 A resolution. Nature. 2009 Aug 20;460(7258):1040-3. Epub 2009 Jul 5. PMID:19578361 doi:10.1038/nature08201
  10. ↑ Gao X, Lu F, Zhou L, Dang S, Sun L, Li X, Wang J, Shi Y. Structure and mechanism of an amino acid antiporter. Science. 2009 Jun 19;324(5934):1565-8. Epub 2009 May 28. PMID:19478139
  11. ↑ Gao X, Zhou L, Jiao X, Lu F, Yan C, Zeng X, Wang J, Shi Y. Mechanism of substrate recognition and transport by an amino acid antiporter. Nature. 2010 Feb 11;463(7282):828-32. Epub 2010 Jan 20. PMID:20090677 doi:10.1038/nature08741
  12. ↑ Fang Y, Jayaram H, Shane T, Kolmakova-Partensky L, Wu F, Williams C, Xiong Y, Miller C. Structure of a prokaryotic virtual proton pump at 3.2 A resolution. Nature. 2009 Aug 20;460(7258):1040-3. Epub 2009 Jul 5. PMID:19578361 doi:10.1038/nature08201
  13. ↑ Casagrande F, Ratera M, Schenk AD, Chami M, Valencia E, Lopez JM, Torrents D, Engel A, Palacin M, Fotiadis D. Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily. J Biol Chem. 2008 Nov 28;283(48):33240-8. Epub 2008 Sep 25. PMID:18819925 doi:10.1074/jbc.M806917200
  14. ↑ Ohhata N, Yoshida N, Egami H, Katsuragi T, Tani Y, Takagi H. An extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4, isolated from crude oil. J Bacteriol. 2007 Oct;189(19):6824-31. Epub 2007 Aug 3. PMID:17675378 doi:10.1128/JB.00872-07
  15. ↑ Reig N, del Rio C, Casagrande F, Ratera M, Gelpi JL, Torrents D, Henderson PJ, Xie H, Baldwin SA, Zorzano A, Fotiadis D, Palacin M. Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters. J Biol Chem. 2007 May 4;282(18):13270-81. Epub 2007 Mar 6. PMID:17344220 doi:10.1074/jbc.M610695200
  16. ↑ Fuchs BC, Bode BP. Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime? Semin Cancer Biol. 2005 Aug;15(4):254-66. PMID:15916903 doi:10.1016/j.semcancer.2005.04.005
  17. ↑ Reig N, del Rio C, Casagrande F, Ratera M, Gelpi JL, Torrents D, Henderson PJ, Xie H, Baldwin SA, Zorzano A, Fotiadis D, Palacin M. Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters. J Biol Chem. 2007 May 4;282(18):13270-81. Epub 2007 Mar 6. PMID:17344220 doi:10.1074/jbc.M610695200
  18. ↑ Bartoccioni P, Del Rio C, Ratera M, Kowalczyk L, Baldwin JM, Zorzano A, Quick M, Baldwin SA, Vazquez-Ibar JL, Palacin M. Role of transmembrane domain 8 in substrate selectivity and translocation of SteT, a member of the L-amino acid transporter (LAT) family. J Biol Chem. 2010 Sep 10;285(37):28764-76. Epub 2010 Jul 7. PMID:20610400 doi:10.1074/jbc.M110.116632

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