Protein unfolding by mitochondria. The Hsp70 import motor

Abstract

Protein unfolding is a key step in the import of some proteins into mitochondria and chloroplasts and in the degradation of regulatory proteins by ATP-dependent proteases. In contrast to protein folding, the reverse process has remained largely uninvestigated until now. This review discusses recent discoveries on the mechanism of protein unfolding during translocation into mitochondria. The mitochondria can actively unfold preproteins by unraveling them from the N-terminus. The central component of the mitochondrial import motor, the matrix heat shock protein 70, functions by both pulling and holding the preproteins.

Fig. 1. The mitochondrial protein import machinery. Shown is the import pathway of a matrix protein. The preprotein is synthesized in the cytosol with an N-terminal positively charged presequence. Cytosolic chaperones can bind to the preprotein. The translocase of the outer mitochondrial membrane (TOM) contains receptors that recognize the presequence and a general import pore (GIP) that mediates translocation across the outer membrane (OM). The translocase of the inner membrane (TIM) includes an import channel formed by Tim23 and Tim17 and the peripheral subunit Tim44. The membrane potential Δψ across the inner membrane (IM) drives translocation of the presequence. Matrix Hsp70 (mtHsp70) binds the preprotein in transit and, together with Tim44 and the co-chaperone Mge1, forms an ATP-dependent import motor. The presequence is cleaved off by the mitochondrial processing peptidase (MPP). IMS, intermembrane space.

Fig. 2. Different unfolding pathways of barnase in solution and by mitochondria. The three portions of barnase are indicated by boxes 1–3 (from N- to C-terminus). In solution, unfolding mainly starts with the middle portion (box 2) of the protein (upper pathway). Mitochondria unfold barnase by unraveling it from its N-terminus (box 1). Barnase was converted to a mitochondrial preprotein by attaching an N-terminal presequence.

Fig. 3. Import driving forces acting on a mitochondrial preprotein. A preprotein has been arrested in the mitochondrial import machinery in a two-membrane spanning manner by attaching a tightly folded domain to the C-terminus [DHFR with bound methotrexate (MTX) that cannot be unfolded by mitochondria]. (A) Two import driving forces, the membrane potential Δψ and matrix Hsp70 (termed Ssc1 in yeast), pull the preprotein in. Therefore, the folded C-terminal domain is tightly pulled against the outer membrane and cannot be cleaved off by proteinase K added to the mitochondria. (B) When both Δψ and mtHsp70 are inactivated, pulling is impaired. The preprotein slides back in the import channel and the C-terminal domain can be cleaved off by proteinase K. (C) A mutant form of mtHsp70 (Ssc1-2) efficiently holds preproteins, but its interaction with the TIM machinery is impaired. Therefore pulling is impaired and, upon dissipation of Δψ, the preprotein slides back. (D) An intragenic suppressor mutation restores the interaction of mtHsp70 with the TIM machinery and thereby restores pulling of the preprotein.