Supplementary material
Fig. S1.
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Fig. S2A.
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Fig. S2B.
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G
Fig. S3.
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References
Cherbas, L. (2004). General procedures for maintenance of Drosophila cell lines.
Indiana University: Drosophila Genomics Resource Center.
Russo, C. A., Takezaki, N. and Nei, M. (1995). Molecular phylogeny and divergence
times of drosophilid species. Mol Biol Evol 12, 391-404.
Rutherford, K., Parkhill, J., Crook, J., Horsnell, T., Rice, P., Rajandream, M. A. and
Barrell, B. (2000). Artemis: sequence visualization and annotation.
Bioinformatics 16, 944-5.
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice. Nucleic
Acids Res 22, 4673-80.
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Supplementary figure captions
Fig. S1. Copper-transporting PIB-type ATPase protein sequence alignment. Human
ATP7A (NP000043.3), human ATP7B (NP000044.2), mouse ATP7A
(NP001103227.1), mouse ATP7B (NP031537.2) and DmATP7 (NP572756.2) were
aligned using a default ClustalW protein sequence alignment (Thompson et al., 1994)
with Boxshade used to highlight conserved residues. The metal binding domains
(GMT/HCxxC), CPC motif and the A (TGE), P (DKTG, TGDN, GDGxND) and N
domains (SEHP) are highlighted (box). The di-leucine, PDZ and FAFDNVGYE
trafficking motifs are also highlighted (box). Residues phosphorylated in human
ATP7A are indicated ().
Fig. S2. Drosophila copper-transporting PIB-type ATPase protein sequence alignment
and phylogenetic analysis. Putative Drosophila P(IB)-type ATPase orthologues were
identified by tBLASTn searches of the genomes available on Flybase
(http://flybase.org/) for sequences similar to DmATP7. Single orthologues were found
for an additional eleven Drosophila species (simulans, sechellia, yakuba, erecta,
ananassae, pseudoobscura, persimilis, willistoni, mojavensis, virilis and grimshawi).
Open reading frames were manually re-annotated using Artemis (Rutherford et al., 2000)
based on sequence similarity with closely related species. (A) Protein sequences were
aligned using ClustalW (Thompson et al., 1994) with Boxshade used to highlight
conserved residues. Each orthologous gene contained four exons and intron splice sites
are indicated (). The conserved CPC motif, A, P and N domains and four metal
binding domains are highlighted (box). The CPC motif could not be identified for D.
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sechellia due to incomplete sequencing in the region. A putative C-terminal di-leucine
motif and Class I PDZ domain (S/TTEL) were conserved in Drosophila orthologues
(box), whereas the ATP7B specific FAFDNVGYE motif was not. Two serine residues
phosphorylated in ATP7A were also conserved in Drosophila (). (B) Phylogenetic
analysis with 100 replicate bootstrapped trees was conducted with the Caenorhabditis
elegans ATP7 used as an out group (fpropars, EMBOSS). The relationship between
these orthologues closely resembles that of the relationship between the species (Russo
et al., 1995). Bioinformatics analysis was conducted using Biomanager 2.0 (Angis, The
University of Sydney, NSW, Australia).
Fig. S3. DmATP7 does not undergo copper-responsive trafficking in Bm3-c2 cells. The
clonal neuronal cell line Dm-BM3-c2, originally isolated from the ventral ganglion of
third instar D. melanogaster larvae were purchased from the Drosophila Genomics
Resource Center (Bloomington, IN, USA). Cells were maintained in M3 media (SigmaAldrich) supplemented with 10% fetal calf serum (Trace Scientific) and 10 µg/ml
insulin (Sigma-Aldrich) at 25C as previously reported (Cherbas, 2004). (A-E)
Immunocytochemical detection of DmATP7 in Drosophila BM3-c2 cells exposed to
basal media (A-C) or media supplemented with 0.8 mM copper for 2.5 h (D-F).
DmATP7 was detected with anti-ATP7B (Green: A, D). Anti-Golgin 97 was used to
detect the TGN (Red: B, E) and DAPI was used to detect the nucleus (Blue). Merged
images are also shown (C, F). Scale bar = 10 µm. DmATP7 partially co-localized with
Golgin 97 at the TGN under both basal and elevated copper conditions. (G) Western
blot detection of DmATP7 in BM3-c2 cells exposed to basal media or media
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supplemented with 0.8 mM copper for 2.5 h. Biotin was used to label cell surface
proteins as described in the materials and methods. Biotinylated samples represent
protein at the cell surface whereas non-biotinylated samples represent intracellular
protein, with total lysate also shown. DmATP7 was detected with anti-ATP7A in the
biotinylated sample demonstrating that this protein is present at the PM, under both
basal and elevated copper conditions.
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