Figure 1.
Phylogenetic and functional characterization of βGPP and α/βHPP.
(A) Bayesian phylogenetic analysis of MPP-like protein sequences using a model [17] that allows for across-tree changes in protein amino acid composition. Scale bar indicates estimated substitutions per site. Posterior probabilities of 1.0 are shown as black dots on nodes, and those greater than 0.95 are shown as values. Bacterial MPP homologues are shown in black, αMPP in red and βMPP in blue. Trichomonas α- and βHPPs and Giardia βGPP are highlighted in green. Only α-proteobacterial relationships are shown for bacteria. The fit between the model and the data is shown in Fig. S1 and the full tree with additional details are shown in Fig. S2. (B) Protein size exclusion chromatography of purified recombinant βGPP showing that it elutes as a single peak between 17 and 44 kDa. The activity of βGPP was assayed for cleavage of the targeting presequence of GiiscU for each fraction and the products were separated by SDS-PAGE. Shift in protein mobility indicates cleavage of a presequence. βGPP activity was only detected in fractions from the central peak. (C) Separation of proteins from a mitosome-rich fraction on a sucrose gradient along with molecular size markers. Bands on the immunoblot and SDS-PAGE were quantified by densitometry. The calculated molecular mass of the βGPP monomer is 44.5 kDa. (D) Processing activity of the αHPP-His (lane 1), βHPP-His (lane 2) and corresponding α/βHPP heterodimer (lane 3) with TviscU, showing that the α- and β-subunits are both required for activity. (E) Specific activities were also determined for the βHPP subunit and the α/βHPP heterodimer with a fluorescent substrate based on the T. vaginalis adenylate kinase presequence (n = 3, mean values with s.d.) The activity of the βHPP subunit by itself is at the limit of detection for this assay.
Figure 2.
Comparative processing of mitosomal, hydrogenosomal and mitochondrial proteins by βGPP, α/βHPP and S. cerevisiae α/βMPP.
The sequence of the demonstrated N-terminal mitochondrial, mitosomal or hydrogenosomal targeting presequences is indicated for each substrate protein with / indicating the cleavage site. Processing of Giardia intestinalis mitosomal presequences (Gifdx, [2Fe2S] ferredoxin; GiiscA and GiiscU, metallochaperones involved in FeS cluster assembly), Trichomonas vaginalis hydrogenosomal presequences (Tvfdx, [2Fe2S] ferredoxin; TvAK, adenylate kinase; Tvhsp70, heat shock protein 70; TviscU, metallochaperone involved in FeS cluster assembly) and mitochondrial presequences (ScMDH, Saccharomyces cerevisiae malate dehydrogenase; MmMDH, Mus musculus MDH; ClMDH, Citrullus lanatus MDH) was tested. Reaction products were separated by SDS-PAGE. Shift in protein mobility indicates cleavage of a targeting presequence. The sites of cleavage indicated by slashes in left column were determined by N-terminal amino acid sequencing. Substrates were incubated with (+) or without (−) the corresponding protease.
Figure 3.
The βGPP is a metallopeptidase with a similar cleavage mechanism to α/βMPP.
(A) Alignment of βGPP and βMPP subunit showing the conserved zinc-binding motif. (B) Effect of protease inhibitors and mutation of E37 on the activity of βGPP. Lane 1: βGPP+GiiscU showing cleavage to produce the mature protein; lane 2: βGPP+GiiscU+serine and cysteine protease inhibitors showing no inhibition; lane 3: βGPP+GiiscU+EDTA showing inhibition of cleavage; lane 4: Mutant βGPP in which E37 was mutated to glutamine+GiiscU, showing that the mutation of a key residue for βMPP activity also eliminates βGPP activity.
Figure 4.
Comparative distribution of charge polarity between mitochondrial, hydrogenosomal and mitosomal peptidases.
(A) Predicted charge polarity distribution of the βHPP subunit and βGPP based on the known structure and charge distribution of the Saccharomyces cerevisiae βMPP subunit [10]. Red and blue colours denote negative and positive charge (±5 kT/e where kT is thermal energy and e is unit charge), respectively, whereas white denote relatively non-polar regions. The yellow asterisk marks the Zn-binding region in the active site of the enzyme (shown in b). The negative charges are distributed evenly in the cavity of βMPP while in the cavity of βGPP the negative charges are concentrated mainly around the active site. The MODELLER program [29] version 9.2 was used to build 3-D models of αHPP, βHPP and βGPP. The electrostatic properties of the model were evaluated using APBS version 0.5.1 [33]. (B) Alignment of key segments where negatively charged residues of βMPP are located and known to interact with the substrate. Numbered residues are those of yeast βMPP. E160 and D164 make a salt bridge with substrate residue R-2 (P2) and F77 interacts with P1′ which is also often a F residue. H70-X-X-E73-H74 is the conserved motif of the active site.