We have identified a complex in mitochondria that functions as a part of the preprotein import machinery of the inner membrane (MIM complex). Two known components, MIM23 and MIM17, and two novel components, MIM33 and MIM14, were found as constituents of this complex. In the presence of a translocating chain, the outer membrane import machinery (MOM complex) and the MIM complex form translocation contact sites. On the matrix side, the MIM complex is associated with the mt-Hsp70-MIM44 system. We propose a structure of the import machinery in which the MIM complex constitutes a proteinaceous channel that accepts preproteins from the MOM complex, facilitates their reversible transmembrane movement, and mediates unidirectional transport by linkage to the ATP-dependent mt-Hsp70-MIM44 system.

Unfolding of preproteins and translocation across the mitochondrial membranes requires their interaction with mt-Hsp70 and Tim44 at the inner face of the inner membrane and ATP as an energy source. We measured the temperature dependence of the rates of unfolding and import into the matrix of two folded passenger domains, the tightly folded heme-binding domain (HBD) of cytochrome b 2 and the loosely folded mouse dihydrofolate reductase (DHFR). Despite the stability of the HBD, its rates of thermal breathing were fast and the preprotein was imported rapidly at all temperatures. In contrast, rates of unfolding and import of DHFR were strongly temperature dependent and import was significantly slower than unfolding. In addition, import rates of DHFR were strongly dependent on the length of the presequence. We propose that the mitochondrial import motor does not exert a constant pulling force. Rather, mt-Hsp70 appears to release a translocating polypeptide chain such that the precursor can then slide back and refold on the surface of the mitochondria. Refolding competes with translocation, and passengers may undergo several rounds of unfolding and refolding prior to their import.

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Determinants in a mitochondrial targeting signal for import and processing were analyzed by introducing deletions into the presequence of cytochrome b,. The matrix targeting signal and the signal recognized by the mitochondrial processing peptidase were found to be separate. The signal for import into the matrix is located at the N-terminus within a stretch of 20 amino acid residues that has the potential to form a positively charged, amphipathic a-helix. The mitochondria1 processing peptidase cleaves after residue 31 and recognizes a short sequence motif around the scissile bond. In the context of a presequence, the cleavage site is accessible for the processing peptidase. At a different location or in a different context, the cleavage site motif is still specifically recognized but processed with lower efficiency. The matrix targeting signal may help to present the cleavage site motif to the mitochondrial processing peptidase.

The incorporation of the gene for cyanamide hydratase can make transgenic tobacco plants resistant to the herbicide cyanamide H2NCN. The herbicide is degraded by the expressed gene to the physiologically harmless urea H2NCONH2. In the presence of the ubiquitous plant enzyme urease, which degrades urea, the hydrolysis product NH can, in fact, serve as nitrogen source. The efficiency of this new approach is impressively demonstrated.