Table 1. Putative substrate proteins identified by mass spectrometry selected for cloning and epitope-tagging, overexpression, and further characterization.

Candidates from the ~37 kDa and ~45 kDa bands were chosen if they were the correct size (± 5 kDa), found exclusively in one band, had a number of unique peptides identified, good peptide coverage of the protein (Supplemental Fig. 5), and were predicted to be phosphorylated (Supplemental Fig. 6). Protein motifs, domains, functions, or homologies are indicated, and were obtained using the NCBI Protein Database at www.ncbi.nlm.nih.gov/protein/. The first column indicates the order in which the protein appeared in the mass spectrometry analysis (lower numbers = larger number of peptides identified). Supplemental Fig. 4 shows peptide locations and coverage. Supplemental Data Set 4 shows all proteins identified by mass spectrometry analysis in the substrate-trapping mutant LMW-PTP pulldown.

#	Identified protein	GenBank accession number(s)	Molecular mass	Location (37 or 45 kDa band)	Number of unique peptides	Percent coverage of protein	Gene size (bp)	Motifs, domains, function, or protein homology
5	Hypothetical protein 328.t00002	XM_644144, XP_649236	34 kDa	37 kDa band	12	36%	915	No conserved domains, matches only proteins in E. histolytica and the related ameba E. dispar. is found in the phagosome and appears to have a role in the regulation of erythrophagocytosis.
6	Type A flavoprotein	XM_651854, XP_656946	46 kDa	45 kDa band	15	44%	1221	Contains a lactamase B domain and a flavodoxin domain. May be involved in detoxifying oxygen and/or nitric oxide.
9	Ser/Thr protein kinase, putative	XM_645070, XP_650162	40 kDa	45 kDa band	9	31%	1065	Is ~50% identical to mammalian STK3/MST2, STK4/MST1, and yeast STE20. MST1 acts as a MAP kinase kinase kinase kinase. STK3/MST2 and STK4/MST1 are involved in caspase activation, apoptosis, and cell proliferation and differentiation.
10	Conserved hypothetical protein gi56465028	XM_001914300, XP_001914335	43 kDa	45 kDa band	7	21%	1041	Belongs to the TLD superfamily (pfam 07534) containing an enzymatic domain, but matches only E. histolytica and E. dispar proteins.
11	ARP2/3 complex 34 kDa subunit, putative	DS571335, EAL49360	34 kDa	37 kDa band	7	27%	882	In mammalian cells, this protein is involved in control of actin polymerization (nucleates new actin strands). Involved in motility and formation of phagocytic cups.
12	Hypothetical protein 503.t00001	XM_650927, XP_656019	43 kDa	45 kDa band	7	19%	1080	Contains a BAR and SH3 domain. BAR domain = involved in dimerization and lipid binding; SH3 domain = binds to proline-rich ligands, expecially PXXP-containing; involved in intramolecular interactions.
18	Ribose-phosphate pyrophospho-kinase, putative	XM_651049, XP_656141	37 kDa	37 kDa band	4	13%	999	Catalyzes the synthesis of phosphoribosyl-pyrophosphate, an intermediate in histidine and tryptophan biosynthesis, and in the de novo biosynthesis of purines and pyrimidines; essential for nucleotide synthesis.