Abstract
The β-barrel protein Tom40 and the α-helically anchored membrane protein Tom22 are the only universally conserved subunits of the protein translocase of the mitochondrial outer membrane (TOM). Tom22 has an N-terminal cytosolic and a C-terminal intermembrane space domain. It occurs in two variants: one typified by the yeast protein which has a cytosolic domain containing a cluster of acidic residues, and a shorter variant typified by the plant protein that lacks this domain. Yeast-type Tom22 functions as a secondary protein import receptor and is also required for the stability of the TOM complex. Much less is known about the more widespread short variant of Tom22, which is also found in the parasitic protozoan Trypanosoma brucei. Here we show that the intermembrane space domain of trypanosomal Tom22 binds mitochondrial precursor proteins and that it is essential for normal growth and mitochondrial protein import. Moreover, complementation experiments indicate that the intermembrane space domain cannot be replaced by the corresponding regions of the yeast or plant Tom22 orthologues. Lack or replacement of the short cytosolic domain, however, does not interfere with protein function. Finally, we show that only the membrane-spanning domain of trypanosomal Tom22 is essential for assembly of the trypanosomal TOM complex analogue.
Highlights
An orthologue of Tom[22], termed Tom[9], has been identified in isolated the mitochondrial outer membrane (TOM) of higher plants[11,12]
ATOM14 is essential for normal growth in both procyclic and bloodstream form cell lines including in an engineered bloodstream form cell line that can grow in the absence of kDNA
Besides ATOM40 which likely is a highly diverged member of the VDAC-like protein family, ATOM14 is the only archaic TOM (ATOM) complex subunit that shows any similarity to components of the TOM complexes of yeast and plants
Summary
An orthologue of Tom[22], termed Tom[9], has been identified in isolated TOM of higher plants[11,12] It has a molecular weight of 9 kDa and has a short cytosolic domain that essentially lacks acidic residues. The amino acid composition of its IMS domain is similar to yeast Tom[22] and is able to functionally replace the corresponding region of the yeast protein[13] It is not known whether plant Tom[9] is essential and whether, as its yeast counterpart, it mediates TOM complex stability. We present an experimental analysis of trypanosomal ATOM14, a remote orthologue of Tom[22], which as the plant-type Tom[22] lacks an acidic cluster in its cytosolic domain. We have analyzed to which extent domains of the yeast and the plant Tom[22] orthologues can function in the context of the trypanosomal protein All these expriments have been done using insect-stage T. brucei
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