Precursor proteins are transported into mitochondria in the absence of proteolytic cleavage of the additional sequences

Many nuclear-coded mitochondrial proteins are synthesized as larger precursor polypeptides that are proteolytically processed during import into the mitochondrion. This processing appears to be catalyzed by a soluble, metal-dependent protease localized in the mitochondrial matrix. In this report we employ an in vitro system to investigate the role of processing in protein import. Intact Neurospora crassa mitochondria were incubated with radiolabeled precursors in the presence of the chelator o-phenanthroline. Under these conditions, the processing of the precursors of the beta-subunit of F1-ATPase (F1 beta) and subunit 9 of the F0F1-ATPase was strongly inhibited. Protease-mapping studies indicated that import of the precursor proteins into the mitochondria continued in the absence of processing. Upon readdition of divalent metal to the treated mitochondria, the imported precursors were quantitatively converted to their mature forms. This processing of imported precursors occurred in the absence of a mitochondrial membrane potential and was extremely rapid even at 0 degrees C. This suggests that all or part of the polypeptide chain of the imported precursors had been translocated into the matrix location of the processing enzyme. Localization experiments suggested that the precursor to F1 beta is peripherally associated with the mitochondrial membrane while the precursor to subunit 9 appeared to be tightly bound to the membrane. We conclude that proteolytic processing is not necessary for the translocation of precursor proteins across mitochondrial membranes, but rather occurs subsequent to this event. On the basis of these and other results, a hypothetical pathway for the import of F1 beta and subunit 9 is proposed.     

Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.     

Mitochondrial protein import. Bypass of proteinaceous surface receptors can occur with low specificity and efficiency

Proteolytic degradation of receptor sites on the mitochondrial surface strongly reduces the efficiency of mitochondrial protein import. The remaining residual import still involves basic mechanisms of protein import, including: insertion of precursors into the outer membrane, requirement for ATP and a membrane potential, and translocation through contact sites between both membranes. The import of a chloroplast protein into isolated mitochondria which occurs with a low rate is not inhibited by a protease-pretreatment of mitochondria, indicating that this precursor only follows the bypass pathway. The low efficiency of bypass import suggests that this unspecific import does not disturb the uniqueness of mitochondrial protein composition. We conclude that mitochondrial protein import involves a series of steps in which receptor sites appear to be responsible for the specificity of protein uptake.     

Plant transformation by microinjection techniques

Several techniques have been developed for introducing cloned genes into plant cells. Vectorless delivery systems such as PEG-mediated direct DNA uptake (e.g. Pasz-kowski et al. 1984), electroporation (e.g. Shillito et al. 1985), and fusion of protoplasts with liposomes (Deshayes et al. 1985) are routinely used in many experiments (see several chapters of this issue). A wide range of plant species, dicotyledonous as well as monocotyledonous, has been transformed by these vectorless DNA transfer systems. However, the availability of an efficient protoplast regeneration system is a prerequisite for the application of these techniques. For cells with intact cell walls and tissue explants the biological delivery system of virulent Agrobacterium species has been routinely used (for review see Fraley et al. 1986). However, the host range of Agrobacterium restricts the plant species, which can be transformed using this vector system. In addition, all these methods depend on selection systems for recovery of transformants. Therefore a selection system has to be established first for plant species to be transformed. The microinjection technique is a direct physical approach, and therefore host-range independent, for introducing substances under microscopical control into defined cells without damaging them. These two facts differentiate this technique from other physical approaches, such as biolistic transformation and macroinjection (see chapters in this issue). In these other techniques, damaging of cells and random manipulation of cells without optical control cannot be avoided so far. In recent years microinjection technology found its application in plant sciences, whereas this technique has earlier been well established for transformation of animal tissue culture cells (Capecchi 1980) and the production of transgenic animals (Brin-ster et al. 1981, Rusconi and Schaffner 1981). Furthermore, different parameters affecting the DNA transfer via microinjection, such as the nature of microinjected DNA, and cell cycle stage, etc, have been investigated extensively in animal cells (Folger et al. 1982, Wong and Capecchi 1985), while analogous experiments on plant cells are still lacking.     

The delta psi- and Hsp70/MIM44-dependent reaction cycle driving early steps of protein import into mitochondria.

New steps in the reaction cycle that drives protein translocation into the mitochondrial matrix have been defined. The membrane potential (delta psi)- and the mtHsp70/MIM44-dependent import machinery cooperate in the transfer of the presequence across the inner membrane. Translocation intermediates, arrested at a stage where only the presequence could form a complex with mtHsp70, still required delta psi for further import. Delta psi at this stage prevented retrograde movement, since mtHsp70 did not bind to the presequence with sufficient affinity. In contrast, mature regions of incoming chains adjacent to the presequence were bound by mtHsp70 tightly enough to stabilize them in the matrix. Cycling of the mtHsp70 on and off incoming chains is a continuous process in the presence of matrix ATP. Both MIM44-bound and free forms of mtHsp70 were found in association with the incoming chains. These data are consistent with a reaction pathway in which the mtHsp70/MIM44 complex acts as a molecular ratchet on the cis side of the inner membrane to drive protein translocation into the matrix.