Coimmunoprecipitation of a GiTom40 partner protein. The Giardia TOM complex was immunoprecipitated from digitonin-solubilized mitosomes with GiTom40 antibodies. Samples of the total cell extract and unbound fractions (corresponding to ∼0.15% of the detergent-solubilized extract) are shown alongside the immunoprecipitated proteins. Immunoglobulin heavy and light chain proteins are indicated (IgG heavy, IgG light), as is GiTom40 and the 32-kDa partner protein. Asterisks indicate minor protein components of the immunoprecipitation that were also analyzed by mass spectrometry.

GiTom40 is found in a hetero-oligomeric complex in mitosomes. (A) Giardia mitosomes (125 μg per lane), or yeast mitochondria (62.5 μg per lane) isolated from a strain expressing GiTom40, were solubilized in digitonin (0.2–4% w/v) and separated by BN-PAGE on 6–16.5% (w/v) polyacrylamide gels (Nijtmans et al. 2002; Wittig et al. 2006). Polyvinylidene difluoride PVDF replicas of the gels were immunodecorated with GiTom40 polyclonal antisera and detected by chemiluminescence. (B) Mitochondria were isolated from yeast expressing GiTom40 and subjected to trypsin shaving (Glick et al. 1992). The samples were analyzed by SDS-PAGE and immunoblotting, using antibodies recognizing GiTom40, the outer membrane protein Tom70, and the intermembrane space protein cytochrome b₂ (Cyb2).

Discovery of a Giardia Tom40 homolog. (A) The mitochondrial targeting β-signal (Kutik et al. 2008) is highlighted in a multiple sequence alignment of GiTom40 and other Tom40 sequences. Shown is a selection of divergent sequences from the 19 used in constructing the HMM. Colors represent amino acids with side chains having similar chemical properties. The β-signal consensus sequence is P_oxGxxH_yxH_yx where P_o represents a polar residue, G is a glycine,and H_y a large hydrophobic residue. (B) CLANS analysis (P value threshold of 10⁻⁸¹) of GiTom40 with 52 Tom40 and 77 VDAC sequences, measuring the relationship between each sequence with all of the others in the analysis. Each point corresponds to the position of one sequence. The specific position of GiTom40 is indicated. (C) Yeast Δtom40 mutants, carrying a counterselectable TOM40 gene and expressing either yeast Tom40 (ScTom40) or GiTom40 were plated onto 5-FOA media to select against the covering plasmid. Cells were incubated for 2 days and only Δtom40 mutants expressing ScTom40 were viable.

GiTom40 is a mitosomal membrane protein. (A) Giardia cell lysate was separated on a nine-step sucrose gradient and 13 (0.9 ml) fractions were collected and analyzed by SDS-PAGE and Coomassie staining (upper panel) or immunoblotting with antisera recognizing GiTom40, the mitosomal protein GiIscS and the endoplasmic reticulum protein GiPDI1. (B) [³⁵S]methionine-labeled precursor proteins were purified from metabolically labeled Escherichia coli (Bradley et al. 1997) and incubated with mitosomes (250 μg protein) at 37 °C. At the indicated time (minutes), aliquots of the reaction mix were removed to ice-cold isotonic buffer, organelles were recovered by centrifugation and resuspended in buffer containing Proteinase K (50 μg/ml) for 15 min at 4 °C. After treatment with PMSF to terminate proteolysis, samples were prepared for SDS-PAGE and the gels analyzed by autoradiography using a Typhoon PhosphorImage scanner (Amersham). “p” refers to the precursor form and “m” the mature, processed form of GiIscU and GiFdx. (C) Giardia intestinalis strain WB was transformed to express the mitosomal protein GiIscU-HA and prepared for immunofluorescence microscopy (Dolezal et al. 2005). 4′,6-Diamidino-2-phenylindole [DAPI]-stained nuclei (blue), mitosomes stained for GiTom40 (red) and GiIscU detected with mouse α-HA (green). The merged image shows the colocalization of GiTom40 with GiIscU.