Mitochondrial protein import - Functional analysis of the highly diverged Tom22 orthologue of Trypanosoma brucei

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.

tRNAs and proteins use the same import channel for translocation across the mitochondrial outer membrane of trypanosomes

“…Depletion of individual ATOM core subunits has variable effects on the architecture of the high molecular weight ATOM complex (32). It has previously been shown that replacement of fulllength ATOM14, the remote Tom22 ortholog of T. brucei, with a truncated variant that lacks the C-terminal intermembrane space (IMS) exposed domain results in a growth arrest and inhibits mitochondrial protein import (45). However, in contrast to the cell line where the entire ATOM14 is depleted, the assembly of the high molecular weight ATOM complex is not affected in the cells expressing the truncated ATOM14 variant (45).…”

“…It has previously been shown that replacement of fulllength ATOM14, the remote Tom22 ortholog of T. brucei, with a truncated variant that lacks the C-terminal intermembrane space (IMS) exposed domain results in a growth arrest and inhibits mitochondrial protein import (45). However, in contrast to the cell line where the entire ATOM14 is depleted, the assembly of the high molecular weight ATOM complex is not affected in the cells expressing the truncated ATOM14 variant (45). We thus asked the question if this protein-import-incompetent ATOM complex is still able to translocate tRNA.…”

“…The ATOM14 3′UTR RNAi cell line and the complementation of this cell line with the truncated ATOM14 lacking the IMS domain is described in ref. 45 Monitoring of Mitochondrial tRNA Abundance. The RNAi cell lines were induced by addition of 1 μg/mL tetracycline to the medium.…”

“…The conditional knockout cell line for ATOM11 was assayed 3 d after removal of tetracycline. Induction times were chosen to coincide with the onset of the growth phenotypes (45). Equal numbers (1-2 × 10 8 ) of uninduced and induced cells were harvested and a mitochondria-enriched fraction was prepared using digitonin extraction and RNase A digestion (30).…”

Mitochondrial protein import in trypanosomes: Expect the unexpected

“…ATOM14 is a remote orthologue of yeast Tom22 that has a much shorter cytosolic domain which, similar to the plant Tom22 orthologue Tom9, lacks the clusters of acidic residues found in the yeast protein . Unlike for Tom22 the short cytosolic domain of ATOM14 is not essential for its function …”