Spreadsheet: c7407a03-35e6-4df6-96dd-c56ab226b61d
Sheet: Sheet1

Supplementary Table 14: Manually curated annotations of T. hominis transport proteins as well as the number of microsporidian homologues in the same cluster. The proteins are annotated according to the [NAME] classification obtained from the TCDB. The description / homologues section gives further details on the manual curation process, functional information on yeast proteins obtained from the yeastdb as well as literature resources, and further descriptions. | Unnamed: 1 | Unnamed: 2 | Unnamed: 3 | Unnamed: 4 | Unnamed: 5 | Unnamed: 6 | Unnamed: 7 | Unnamed: 8 | Unnamed: 9
short name | TC | TCDB family | T hom | N cer | E cun | E int | T. hominis proteins: | human/yeast homologue in cluster? | description / homologues
ABC (TM-NBD) | 3.A.1 | The ATP-binding Cassette (ABC) Superfamily | 3 | 6 | 5 | 6 | orf_2794, orf_2378, orf_2384 | y | The only yeast transporters with this domain conformation are mitochondrial Atm1, Mdl1 &  Mdl2 and two peroxisomal transporters Pxa1 & Pxa2 involved in fatty acid oxidation. BLAST analyses of the T. hominis proteins against the TCDB, recovered yeast Atm1 as the best hit for two of the three proteins.
ABC (NBD-TM) | 3.A.1 | The ATP-binding Cassette (ABC) Superfamily | 7 | 4 | 5 | 6 | orf_902, orf_57, orf_1710, orf_2212, orf_3158, orf_2443, orf_3169 | y | Based on BLAST results these sequences belong to the ABCG superfamily of ABC transporters, which has been implicated in sterol and lipid import as well as toxin export.
MFS (a) | 2.A.1 | The Major Facilitator Superfamily (MFS) | 4 | 1 | 2 | 2 | orf_1192, orf_1681, orf_3170, orf_963 | y | Some indications point to a function as sugar transporters - best BLAST hits are mostly bacterial sequences, which sometimes have a sugar transport domain, e.g. for maltose. The E. intestinalis protein has a domain for nucleoside/xanthine transport, orf_963 has a conserved domain (CDD) annotated as a sugar efflux transporter and phenyl propionate permease family.
MFS (b) | 2.A.1 | The Major Facilitator Superfamily (MFS) | 1 | 1 | 1 | 1 | orf_3171 | y | Orf_3171 was assigned to a KOG as a predicted sugar transporter. BLAST analysis against the TCDB recovers an unknown MFS transporter as best hit.
MFS (c) | 2.A.1 | The Major Facilitator Superfamily (MFS) | 2 | 2 | 2 | 2 | orf_3146, orf_315 | y | The proteins orf_3146 and orf_315 encode the [NAME] domain PF00083 which is annotated as a sugar/inositol transporter. The respective transporter database TCDB entry 2.A.2. is annotated as a general sugar MFS transporter where transport substrates are mostly glycosides but also other sugars. The Microsporidia sequences of this cluster group together with two yeast proteins, which are members of the sugar porter family. BLAST analysis against TCDB recovers a glucose MFS transporter and other hexose MFS transporters with high support.
MFS (d) | 2.A.1 | The Major Facilitator Superfamily (MFS) | 1 | 1 | 1 | 1 | orf_2234 | n | The sequences of E. cuniculi and E. intestinalis show a short hit to a folate transporter domain (CDD) but no evidence for that in the T. hominis protein so it is unclear whether it acts as a folate transporter. BLAST analysis against TCDB shows that the T. hominis orf is a highly divergent  MFS proteinwith no clear affiliation to any group.
MFS (e) | 2.A.17 | The Proton-dependent [NAME] (POT) Family | 1 | 1 | 1 | 1 | orf_2975 + orf_2976 | n | The proteins orf_2975+2976 (frameshift), [NAME], C4V8Y0_NOSCE, E0S9X0_ENCIT do not form a cluster but show similarities to [NAME] domains for MFS proteins. All four proteins shows similarity to an oligopeptide transporter domain. BLAST analysis against the TCDB recovers the proton-dependent oligopeptide transporter family first, which is a member of the MFS superfamily.
NTT | 2.A.12 | The ATP:ADP Antiporter (AAA) Family | 4 | 4 | 4 | 4 | orf_2860, orf_2894, orf_2896, orf_1166 | n | Nucleotide transport, energy aquisition (ATP, possibly other nucleotides)
CPA1 | 2.A.36 | The Monovalent Cation:Proton Antiporter-1 (CPA1) Family | 2 | 1 | 1 | 1 | orf_436, orf_2933 | y | The S. cerevisiae protein in the cluster is described as a Na+/H+ and K+/H+ exchanger, which is required for intracellular sequestration of Na+ and K+; this protein is located in the vacuole and late endosome compartments and is required for osmotolerance to acute hypertonic shock and for vacuolar fusion. Strong similarity of microsporidian proteins to proteins with this function are also supported by BLAST analyses.
CaCA | 2.A.19 | The Ca2+:Cation Antiporter ([NAME]) Family | 1 | 1 | 1 | 1 | orf_2669 | n | The best BLAST hit is an Arabidopsis thaliana sodium/calcium exchanger family protein which is described as located in the endomembrane system; all other proteins are similarly annotated. BLAST against TCDB recovers the calcium/cation exchange family as best hits.
V-ATPase (a) | 3.A.2 | The H+- or Na+-translocating F-type, V-type and A-type ATPase (F-ATPase) Superfamily | 3 | 2 | 3 | 3 | orf_529, orf_2367, orf_1792 | y | Three different S. cerevisiae proteins are in this cluster: Cup5p is a proteolipid subunit of the vacuolar H(+)-ATPase V0 sector (subunit c; dicyclohexylcarbodiimide binding subunit), this protein is required for vacuolar acidification and is important for copper and iron metal ion homeostasis. Ppa1p is a cytoplasmic inorganic pyrophosphatase (PPase), it forms a homodimer that catalyzes the rapid exchange of oxygens from Pi with wate. Tfp3p is a vacuolar ATPase V0 domain subunit c, it is involved in proton transport activity and both N and C termini are in the vacuolar lumen. Results from the phylogenetic analysis show that orf_1792 clusters with the yeast protein Ppa1. Orf_529 and orf_2367 both cluster with the other microsporidian species but not with the other two yeast sequences.  Orf_529 and orf_2367 may function as V-ATPases, but their specific role is not clear.
V-ATPase (b) | 3.A.2 | The H+- or Na+-translocating F-type, V-type and A-type ATPase (F-ATPase) Superfamily | 1 | 1 | 1 | 1 | orf_580 | y | Two different S. cerevisiae proteins are in this cluster: Stv1p and Vph1p are subunits of the vacuolar-ATPase V0 domain, and represent two isoforms. Stv1p is located in V-ATPase complexes of the Golgi and endosomes while Vph1p is located in V-ATPase complexes of the vacuole. A specific relationship of the microsporidian sequences to either of these two yeast proteins was not recovered in phylogenetic analyses; the microsporidian proteins are likely to function as V-ATPases but their location(s) in the endomembrane system are not not resolved.
P-ATPase (a) | 3.A.3 | The P-type ATPase (P-ATPase) Superfamily | 6 | 4 | 4 | 4 | orf_542, orf_2789 (+2788 frameshift), [NAME], orf_2279, orf_2150, orf_1801 | y | These proteins are in a cluster with 14 S. cerevisiae proteins. In the phylogenetic analysis, orf_2150 and one sequence from each of the other Microsporidia species groups with one human protein and Spf1 from yeast, which is a P-type ATPase and functions as an ion transporter of the ER membrane and is involved in ER function and Ca2+ homeostasis. orf_542  and one sequence from each of the other Microsporidia (except E. bieneusi, where 3 homologues group here) do not form a group with yeast directly but has a common ancestor with orf_2150, Spf1, and Vpk9. The latter is a vacuolar protein with a possible role in sequestering heavy metals; it has similarity to the type V P-type ATPase Spf1p. [NAME] and one sequence from each of the other Microsporidia groups with neo1 from yeast; this protein confers neomycin resistance, it is a putative aminophospholipid translocase (flippase) involved in endocytosis and vacuolar biogenesis and localizes to endosomes and the Golgi aparatus. orf_1801 and one sequence from each of the other Microsporidia group with several human and four yeast proteins where all four are aminophospholipid translocases (flippase); two of these proteins (dnf3 and dsr2) are trans-golgi network vesicle located, two (dnf1 and dnf2) are plasma membrane located, and are involved in endocytosis. orf_2789 is the only Microsporidia sequence to cluster with yeast pmc1, which is a vacuolar Ca2+ ATPase involved in depleting cytosol of Ca2+ ions; this prevents growth inhibition by the activation of calcineurin in the presence of elevated concentrations of calcium and is similar to the mammalian PMCA1a. orf_2279 branches off on its own with no other Microsporidia sequences clustering together.
P-ATPase (b) | 3.A.3 | The P-type ATPase (P-ATPase) Superfamily | 1 | 1 | 0 | 0 | orf_1969 | y | These proteins, where no Encephalitozoon homologues could be identified, cluster with the S. cerevisiae proteins Pca1p and Ccc2p. Pca1 is a cadmium transporting P-type ATPase; and it may also have a role in copper and iron homeostasis. Ccc2 is a Cu(+2)-transporting P-type ATPase, and is required for export of copper from the cytosol into an extracytosolic compartment. orf_1969 clusters with Pca1 but onlyweakly, and is therefore likely to function like either of these two yeast proteins, but it is unclear which one.
GMA | 2.A.7.13 | The GDP-Mannose:GMP Antiporter (GMA) Family | 1 | 2 | 2 | 2 | orf_1491 | y | These proteins are in a cluster with the S. cerevisiae protein Vrg4, which is a Golgi GDP-mannose transporter which regulates Golgi function and glycosylation in Golgi. This is consistent with a mannosylation pathway being present in T. hominis. Two other S. cerevisiae proteins, Hvg1 and Ymd8 are in the same cluster; these have an unknown function but are described as similar to Vrg4. The T. hominis protein is very divergent and did not hit any protein in the TCDB; the putative functional classification was based on the yeast protein Vrg4.
N/S | 2.A.7 | The Drug/Metabolite Transporter (DMT) Superfamily | 2 | 1 | 1 | 1 | orf_792, orf_799 | y | orf_799 encodes a general transporter domain; this cluster includes a human nucleotide-sugar transporter, which is assumed to be the function of these proteins. BLAST analysis against TCDB showed high divergence of the T. hominis resulting in inconsistent hits; the classification is based on the human protein within the cluster.
CDF | 2.A.4 | The Cation Diffusion Facilitator (CDF) Family | 1 | 1 | 1 | 1 | orf_3135 | y | These proteins are in a cluster with a S. cerevisiae zinc transporter, which are both annotated to transport zinc into vacuoles for storage.
Zn2+ (a) | 2.A.5 | The Zinc (Zn2+)-Iron (Fe2+) Permease (ZIP) Family | 4 | 1 | 1 | 1 | orf_1424, orf_2017, orf_2019, orf_2026 | y | These proteins are in a cluster with the S. cerevisiae zinc transporters zrt1 and [NAME], which are both described as plasma-membrane located.
Zn2+ (b) | 9.B.30 | [NAME] (Hly III) Family | 1 | 2 | 1 | 1 | orf_1901 | y | These proteins cluster together with the S. cerevisiae proteins Izh1, 2 and 4: IZH1, IZH2, IZH3, and IZH4 encode a family of paralogous membrane proteins thought to affect zinc homeostasis either by altering membrane sterol content or by directly altering cellular zinc levels. The [NAME] proteins belong to a large and nearly ubiquitous family of proteins found in both prokaryotes and eukaryotes. Three possible functions have been proposed for the [NAME] proteins; these proteins may function solely in sterol metabolism by influencing the permeability of the plasma membrane and, consequently, the homeostasis of cations such as zinc; or the Izh proteins function as transporters for zinc used in a signaling capacity, a possibility that may explain their regulation by Zap1p and their effect on Zap1p activity; or the [NAME] proteins are involved in a signal transduction cascade that is independent of zinc, and that Zap1p is a downstream target of this pathway.
MscS (a) | 1.A.23 | The Small Conductance Mechanosensitive Ion Channel (MscS) Family | 4 | 4 | 3 | 3 | orf_524, orf_1684, orf_1618, orf_2792 | n | Small conductance ion channels provide protection against osmotic shock and are found in all 3 domains of life. Despite the lack of yeast or human proteins in these clusters, the functional annotation is clearly supported by BLAST analyses as well as domains encoded on these proteins.
MscS (b) | 1.A.23 | The Small Conductance Mechanosensitive Ion Channel (MscS) Family | 2 | 1 | 1 | 1 | orf_527, orf_1619 | n | Small conductance ion channels provide protection against osmotic shock and are found in all 3 domains of life. Despite the lack of yeast or human proteins in these clusters, the functional annotation is clearly supported by BLAST analyses as well as domains encoded on these proteins.
MscS (c) | 1.A.23 | The Small Conductance Mechanosensitive Ion Channel (MscS) Family | 1 | 1 | 1 | 1 | orf_1922 | n | Small conductance ion channels provide protection against osmotic shock and are found in all 3 domains of life. Despite the lack of yeast or human proteins in these clusters, the functional annotation is clearly supported by BLAST analyses as well as domains encoded on these proteins.
Mg2+ (a) | 1.A.35 | The CorA Metal Ion Transporter (MIT) Family | 1 | 1 | 0 | 0 | orf_1143 | y | This cluster does not contain any Encephalitozoon sequences; the proteins from T. hominis and N. ceranae cluster with 2 yeast proteins which are both described to function as plasma membrane [NAME]) transporters. The protein expression and turnover are regulated by [NAME]) concentration; an overexpression confers increased tolerance to [NAME]+) and [NAME]+) ions in yeast.
Mg2+ (b) | 9.A.19 | The Mg2+ Transporter-E (MgtE) Family | 1 | 1 | 1 | 1 | orf_1647 | y | These proteins cluster with human magnesium plasma membrane transporters, which is therefore their most likely function.
Mg2+ (c) | 2.A.7 | The Drug/Metabolite Transporter (DMT) Superfamily | 1 | 1 | 1 | 1 | orf_700 | y | These proteins clusters with a human Mg2+ transporter, which can also transport other divalent cations such as Ba2+, Mn2+, Sr2+ and Co2+ but to a much less extent than Mg2+. This protein gets recruited to the cell membrane in response to low extracellular magnesium; it is annotated to be early endosomal but this annotation is only based on similarity.
Cu+ | 1.A.56 | The Copper Transporter (Ctr) Family | 1 | 1 | 0 | 0 | orf_2688 | n | The T. hominis protein but not the N. ceranea protein, shares some similarity to copper transporters; however, it is missing the MXXXM motif described as essential in Puig et al. Journal of Biological Chemistry. 2002 Jul;277(29):26021-30.
SulP | 2.A.53 | The Sulfate Permease (SulP) Family | 2 | 1 | 1 | 1 | orf_2963, orf_2965 | y | The S. cerevisiae protein in this cluster is annotated as unknown function; however, orf_2963 and orf_2965 have similarity to the SulP domain PF00916. This is a large family of transport proteins with members in archaea, bacteria, fungi, plants, animals, which can transport sulfate but also other anions; often one transporter can perform several functions; see Ohana et al. J [NAME]. 2011 Feb;137(2):239-51 for a recent description of their transport potential.
Pi | no entry found | no entry found | 1 | 1 | 1 | 1 | orf_2938 | y | The S. cerevisiae protein in this cluster has been functionally characterized as inorganic phosphate transporter; a disruption and high dosage leads to reduced Pi uptake.
AAAP | 2.A.18 | The Amino Acid/Auxin Permease (AAAP) Family | 2 | 7 | 6 | 6 | orf_2432, orf_2314 | y | The proteins in theis cluster show strong similarity to proteins of the AAAP family and encode the motif PF01490. Proteins of the AAAP family (TCDB 2.A.18) can transport auxin (indole-3-acetic acid), a single amino acid or multiple amino acids. Some of the permeases are described to have a very broad specificity transporting several amino acids.
MIP | 1.A.8 | The Major Intrinsic Protein (MIP) Family | 1 | 1 | 1 | 1 | [NAME] | y | This cluster contains a yeast aquaporin - aquaporins have been described in Microsporidia to function as spore-specific water channels.
CTL | 2.A.92 | The [NAME]-like (CTL) Family | 1 | 3 | 1 | 1 | orf_1213 | y | This protein appears to contain a choline-transporter domain; however, the yeast protein within this cluster has the same domain but no apparent function in choline transport in [NAME]. Neurochem Res. 2004 Feb;29(2):461-7, so it is uncertain if the microsporidian proteins function in choline transport.
 |  |  |  |  |  |  |  |  | clusters with unknown functions
unknown (a) |  |  | 1 | 1 | 1 | 1 | orf_6 | n | Based on HHPRED - similarity to PF03661 UPF0121:  Uncharacterised protein family (UPF0121);  InterPro: IPR005344 Uncharacterised integral membrane protein family.; GO: 0016021 integral to membrane. Therefore likely to be in the membrane, possibly transporter, but function unknown
unknown (b) |  |  | 1 | 1 | 1 | 1 | orf_44 | n | No similarities could be recovered based on HHPRED.
unknown (c) |  |  | 1 | 0 | 1 | 1 | orf_1513 | n | HHPRED hits PF00335 tetraspannin which is a membrane-bound protein involved in cell-cell interactions; so it is unclear whether these proteins actually function as transporters. orf_1513 was recovered in our spore proteomics data.
unknown (d) |  |  | 1 | 1 | 1 | 1 | orf_2446 | n | No similarities could be recovered based on HHPRED.
unknown (e) |  |  | 2 | 1 | 1 | 1 | orf_2882, orf_117 | n | No similarities could be recovered based on HHPRED.
unknown (f) |  |  | 8 | 0 | 2 | 2 | orf_1334, orf_1382, orf_150, orf_257, orf_3244, orf_51, orf_52, orf_53 | n | No similarities could be recovered based on HHPRED.
unknown (g) |  |  | 1 | 1 | 2 | 2 | orf_522 | y | The S. cerevisiae protein in this cluster, as well as hits recovered by BLAST, are described as membrane proteins of unknown function. Orf_522 was recovered in our spore proteomics data.
unknown (h) |  |  | 2 | 1 | 1 | 1 | orf_2715, orf_2716 | n | No similarities could be recovered based on HHPRED.
unknown (i) |  |  | 1 | 1 | 1 | 1 | orf_979 | n | HHPRED search hits PTHR14856, a domain of unknown function, and shows some similarity to a PQ loop repeat family protein domain; the PQ loop is a general domain found between transmembrane segments and can function in lysosomal targeting, but also has other unknown functions.
unknown (j) |  |  | 1 | 1 | 1 | 1 | orf_2878 | n | No similarities could be recovered based on HHPRED.
 |  |  |  |  |  |  |  |  | T. hominis singletons with putative functional annotations
GMA | 2.A.7.13 | The GDP-Mannose:GMP Antiporter (GMA) Family |  |  |  |  | orf_2291 |  | The T. hominis protein encodes for a large insertion, but the surrounding areas recover Microsporidia and other sequences by BLAST search; based on similarities this is most likely another Golgi GDP-mannose transporter.
AE | 2.A.31 | The Anion Exchanger (AE) Family |  |  |  |  | orf_1222 |  | Based on BLAST hits, the recovered proteins are mainly annotated as anion exchange transporters; this protein also encodes the [NAME] domain PF00955 annotated as HCO3- cotransporter (CL-/HCO3-).
 |  |  |  |  |  |  |  |  | Putative T.hominis-specific transporters identified by the presence of multiple transmembrane domains (TMDs)
 |  |  |  |  |  |  | orf_267 |  | No above-threshold hits with PSI-BLAST, 10 TMDs
 |  |  |  |  |  |  | orf_258 |  | No above-threshold hits with PSI-BLAST, possibly 3-4 TMDs
 |  |  |  |  |  |  | orf_744 |  | No above-threshold hits with PSI-BLAST, 8 TMDs
 |  |  |  |  |  |  | orf_1202 |  | No above-threshold hits with PSI-BLAST, 6 TMDs
 |  |  |  |  |  |  | orf_1238 |  | No above-threshold hits with PSI-BLAST, 6 TMDs
 |  |  |  |  |  |  | orf_1645 |  | No above-threshold hits with PSI-BLAST, 8 TMDs
 |  |  |  |  |  |  | orf_1881 |  | No above-threshold hits with PSI-BLAST, 5 TMDs
 |  |  |  |  |  |  | orf_1886 |  | No above-threshold hits with PSI-BLAST, 7 TMDs. orf_1886 was recovered in our proteomics data.
 |  |  |  |  |  |  | orf_1941 |  | No above-threshold hits with PSI-BLAST, 10 TMDs
 |  |  |  |  |  |  | orf_2673 |  | Similarity to an unknown protein, 7 TMDs
 |  |  |  |  |  |  | orf_2823 |  | No above-threshold hits with PSI-BLAST, 7 TMDs
 |  |  |  |  |  |  | orf_2903 |  | No above-threshold hits with PSI-BLAST, 11 TMDs
 |  |  |  |  |  |  | orf_2491 |  | No above-threshold hits with PSI-BLAST, possibly 3-5 TMDs
 |  |  |  |  |  |  | orf_805 |  | No above-threshold hits with PSI-BLAST, possibly 3-4 TMDs
 |  |  |  |  |  |  | orf_469 |  | No above-threshold hits with PSI-BLAST, possibly 2-4 TMDs
 |  |  |  |  |  |  | orf_515 |  | No above-threshold hits with PSI-BLAST, 7 TMDs
 |  |  |  |  |  |  | orf_806 |  | No above-threshold hits with PSI-BLAST, 4 TMDs
 |  |  |  |  |  |  | orf_2793+orf_2792 |  | Frameshift, using the full-length sequence recovers only Microsporidia proteins, all annotated as hypotheticals., 4 TMDs
 |  |  |  |  |  |  | orf_3282 |  | No above-threshold hits with PSI-BLAST, 5 TMDs
 |  |  |  |  |  |  | orf_775 |  | Shows similarity to a eukaryotic membrane protein present in many eukaryotes but no other [NAME] of unknown function, 5 TMDs
 |  |  |  |  |  |  | orf_673+orf_674 |  | Widely conserved hypothetical membrane protein (singleton due to frameshift, which caused failure of the length restriction limitation), 7 TMDs
 |  |  |  |  |  |  | orf_1885 |  | No above-threshold hits with PSI-BLAST. [NAME] was recovered in our spore proteomics data, 5 TMDs
 |  |  |  |  |  |  | orf_2523 |  | No above-threshold hits with PSI-BLAST, 6 TMDs
 |  |  |  |  |  |  | orf_2680 |  | No above-threshold hits with PSI-BLAST, 4 TMDs
 |  |  |  |  |  |  | orf_1649 |  | No above-threshold hits with PSI-BLAST, 6 TMDs
 |  |  |  |  |  |  | orf_1347 |  | No above-threshold hits with PSI-BLAST, 8 TMDs
 |  |  |  |  |  |  | orf_2931 |  | No above-threshold hits with PSI-BLAST, 8 TMDs
 |  |  |  |  |  |  | orf_2806 |  | No above-threshold hits with PSI-BLAST, 7 TMDs
 |  |  |  |  |  |  | orf_1567 |  | No above-threshold hits with PSI-BLAST, 9 TMDs
 |  |  |  |  |  |  | orf_702 |  | BLAST search shows similarity to Plasmodium sp. unknown proteins. PSI-BLAST also recovers a bacterial ABC transporter, 6 TMDs
 |  |  |  |  |  |  | orf_3082 |  | No above-threshold hits with PSI-BLAST, 6 TMDs
 |  |  |  |  |  |  | orf_3119 |  | No above-threshold hits with PSI-BLAST, possibly 2-4 TMDs
 |  |  |  |  |  |  | orf_761 |  | No above-threshold hits with PSI-BLAST, 6 TMDs