Figure 1: Sub-cellular localization of T. hominis Fe/S-cluster assembly components.

Thawed cryo-sections of glutaraldehyde-fixed T. hominis-infected RK-13 cells were labelled with antibodies to T. hominis ISC components and protein-A gold. (a) Quantitative analysis. Labelling is expressed as density of gold labelling over compartment profile area (estimated by point counting; see ‘Methods’ section; 34–40 micrographs analysed per protein/experiment with following number of golds per experiment: ThIsu1, 64; ThNfs1, 67; ThYah1, 35; ThYfh1, 70; ThIsd11, 111). Error bars indicate the s.e.m. (N=3). (b) Images illustrating the distribution of labelling for ISC components in mitosomes (these show examples of positive labelling only and therefore do not reflect densities quantified in a). Labelling appears to be located over the matrix of the double membrane-bounded organelle profiles (with mean minor and major axes of 80 and 127 nm, respectively; N=50 mitosome profiles). The analysed profiles were morphologically undistinguishable from mitosomes that labelled positively for ThHsp70 as shown here and described previously³. Bars=100 nm. (c) The distribution of immunogold labelling for the indicated ISC proteins over mitosome profiles was compared with an equivalent number of random points. (IM, inner mitosomal membrane location). Labelling was expressed as gold particles per random point count and indicates the concentration of labelling at the IM/matrix interface. The numbers of gold and random counts were as follows: ThIsu1, 79; ThNfs1, 66; ThYah1, 37; ThYfh1, 63; ThIsd11, 50. Labelling for all tested ISC components was towards the mitosomal matrix with an enrichment at the IM. (d) Immunogold labelling distribution over mitosome matrix. Cumulative fraction plot shows pooled gold labelling data for all five ISC components (ISC labelling) compared with points located simple uniform random (random). ISC components show relative enrichment of labelling close to the IM (see Supplementary Fig. 3 for individual analyses).