Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life
1 Department of Molecular Biology, Aarhus University, C. F. Møllers Allé 3, Aarhus C, DK-8000, Denmark
2 Institute of Clinical Medicine, Aarhus University, Brendstrupgårdsvej 100, Aarhus N, DK-8200, Denmark
3 Department of Haematology, Aarhus University Hospital, Tage-Hansens gade 2, DK-8000 Aarhus C, Denmark
4 Department of Medical Biochemistry, Ole Worms Allé 3, Aarhus University, DK-8000 Aarhus C, Denmark
BMC Biochemistry 2011, 12:21 doi:10.1186/1471-2091-12-21Published: 17 May 2011
Additional file 1:
Protein sequence alignment of nine PiT family members from all kingdoms. A The 10 putative TM domains according to the Johann topology model are shown on the human PiT2 sequence using black boxes with white filling [8,20]; the putative large intracellular domain (L6) of human PiT2, according to this model, spans the amino acid sequence: P236-V483. The N-terminal and C-terminal PiT family signature sequences  are shown on the alignment in black boxes with grey filling. Human PiT1 E70 in the 2nd TM domain and human PiT2 H502 in the 7th TM domain are indicated with circles. The TMHMM-predicted TM domains of the eukaryotic protein sequences for PiT family members and the DAS-predicted TM domains of the prokaryotic protein sequences for PiT family members are shown in black bold. The red bold sequences represent TM-domains, which we suggest exist, however, they were not predicted by the servers: N. crassa Pho-4+ TM 1 (sequence Q5-I24) is suggested to be homologous to the TM 1 predicted in the C. elegans putative phosphate permease protein sequence. The presence of Pho-4+ TM 1 is also based on the assumption that the N-terminal PiT-family signature sequences should be placed equivalently (extracellularly in L1) in all PiT family members. A. thaliana Pht2_1 TM 2 (sequence A187-G211) is suggested to be homologous to the TM 2 predicted in the T. brucei putative phosphate permease protein sequence. The presence of Pht2_1 TM 2 is also based on experimental assignment of the L6 for rat PiT2 to the cytoplasmic space , and Pht2_1 therefore requires a TM 2 to fulfill this criteria. H. sapiens PiT2 TM 3 (sequence T83-A105) is suggested to be homologous to the TM 3 predicted in the H. sapiens PiT1 protein sequence. Investigation of a human PiT1/PiT2 chimera where the PiT1 backbone harbors the human PiT2 sequence G120-V141 showed that this sequence conferred A-MLV receptor function upon human PiT1 , and the G120-V141 sequence is therefore highly likely extracellular in both human PiT paralogs and this requires the presence of TM 3 in human PiT2. TM 7 domains in putative phosphate permeases from C. elegans (sequence Q330-A349), D. melanogaster (sequence M472-G491), T. brucei (sequence Y346-A365), and N. crassa Pho-4+ (sequence Y318-A337) are suggested to be homologous to the TM 7 predicted in H. sapiens PiT2 and PiT1 sequences. The presence of TM 7 in putative phosphate permeases from C. elegans, D. melanogaster, T. brucei, and N. crassa Pho-4+ is also based on the assumption that the C-terminal PiT-family signature sequences should be placed equivalently (extracellularly in L7) in all PiT family members. Moreover, investigation of a Pho-4+/human PiT2 chimera where the Pho-4+ backbone harbors the human PiT2 sequences C117-I143 (stretch in L3) and L512-A531 (stretch in L7) showed that these sequences confer A-MLV receptor function upon Pho-4+, and these sequences are therefore highly likely extracellular and this requires the presence of a TM 7. Similarly, investigation of a Pho-4+/human PiT1 chimera where the Pho-4+ backbone harbors the human PiT1 sequence L545-S556 (stretch in L7) showed that these sequences confer GALV receptor function upon Pho-4+. H. sapiens PiT2 TM 9 (sequence G571-S593) and H. sapiens PiT1 TM 9 (sequence G599-S521) are suggested to be homologous to the TM 9 predicted in RPHO-1 R. norvegicus (human PiT1 ortholog) protein sequence G601-S623 [Swiss-Prot:Q9JJP0] using the TMHMM server (data not shown). N. crassa Pho-4+ TM 9 (sequence L523-G545) is suggested to be homologous to the TM 9 predicted in C. elegans putative phosphate permease protein sequence. The TM 9 is required to orient the TM 10 equivalently in all PiT family members. Lower case letters represent TMHMM- or DAS-predicted TM sequences, which we based on either too small length to comprise a TM or due to suggested extracellular position (see above) found were non-compatible with regular TM domains; however, these sequences might instead "dip" into the membrane lipid bilayer. It should be noted that these sequences are counted as being part of the loop sequences in Figure 4. A star (✰) (labeled a to i) and a vertical line indicate the position of an intron-exon border in each of the human PiT genes determined by use of the SPIDEY mRNA-to-genome DNA alignment as described in "Methods". Below the alignment, the names, species, phylas, kingdoms, Swiss-Prot accession numbers, and the amino acid lengths of the nine proteins are given. B The server-predicted TM domains (black boxes) and the by us suggested TM domains (red boxes) for each of the nine PiT family members are depicted in order to illustrate the conservedness of the TM domains: TM 4, TM 8, TM 10 (fully conserved) > TM 5, TM 6 (fully conserved in eukaryotes) > TM 1, TM 2, TM 3 > TM 9 > TM 7 (least conserved). The white asterisk indicates a prediction of a unique TM domain in the unusually long N-terminal sequence of A. thaliana Pht2_1.
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Additional File 2:
Data and statistics. Average 32Pi uptakes in oocytes given as pmol/oocyte-hour ±SEM, information regarding the number (n) of oocytes measured, and the statistics (P values) for Figures 3A-B and Figure 6 are available in Additional File 2. Average numbers of blue (infected) cells per dish from three dishes ±SEM and the statistics (P values) for Figures 3C-E are available in Additional File 2. Average loop lengths given as amino acids ±SEM and information regarding the number (n) of loops counted for Figure 4B are available in Additional File 2.
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