Abstract
The mitochondrial import receptor Tom70 contains a tetratricopeptide repeat (TPR) clamp domain, which allows the receptor to interact with the molecular chaperones, Hsc70/Hsp70 and Hsp90. Preprotein recognition by Tom70, a critical step to initiate import, is dependent on these cytosolic chaperones. Preproteins are subsequently released from the receptor for translocation across the outer membrane, yet the mechanism of this step is unknown. Here, we report that Tom20 interacts with the TPR clamp domain of Tom70 via a conserved C-terminal DDVE motif. This interaction was observed by cross-linking endogenous proteins on the outer membrane of mitochondria from HeLa cells and in co-precipitation and NMR titrations with purified proteins. Upon mutation of the TPR clamp domain or deletion of the DDVE motif, the interaction was impaired. In co-precipitation experiments, the Tom20-Tom70 interaction was inhibited by C-terminal peptides from Tom20, as well as from Hsc70 and Hsp90. The Hsp90-Tom70 interaction was measured with surface plasmon resonance, and the same peptides inhibited the interaction. Thus, Tom20 competes with the chaperones for Tom70 binding. Interestingly, antibody blocking of Tom20 did not increase the efficiency of Tom70-dependent preprotein import; instead, it impaired the Tom70 import pathway in addition to the Tom20 pathway. The functional interaction between Tom20 and Tom70 may be required at a later step of the Tom70-mediated import, after chaperone docking. We suggest a novel model in which Tom20 binds Tom70 to facilitate preprotein release from the chaperones by competition.
Highlights
Tom70 is a member of the tetratricopeptide repeat (TPR) co-chaperone family characterized by a specialized dicarboxylate TPR clamp domain that interacts with molecular chaperones, Hsp70/Hsc70 and/or Hsp90
We hypothesized that the 100-kDa adduct may be an internal cross-link of the monomeric Tom70, or it may represent a heterodimeric crosslink with a smaller component of the outer mitochondrial membrane
In the sequence alignment of the C terminus of Tom20, we found that human Tom20 ends with a DDVE sequence that is very similar to the EEVD motif at the C terminus of the chaperones Hsc70/Hsp70 and Hsp90 (Fig. 1A)
Summary
Tom70 is a member of the tetratricopeptide repeat (TPR) co-chaperone family characterized by a specialized dicarboxylate TPR clamp domain that interacts with molecular chaperones, Hsp70/Hsc70 and/or Hsp90. We first created a C-terminal deletion mutant of Tom20 that lacks the four-residue DDVE motif (Tom20⌬C) and compared it with its full-length counterpart by cross-linking to mitochondrial outer membrane proteins (Fig. 2B). We conclude that the Tom20-Tom70 interaction likely requires the DDVE motif and the TPR clamp domain in the respective proteins on the membrane.
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