Translocation of precursors

Import Receptors and Membrane Translocation of Precursor Peptides 139... 

Bolliger L, Junne T, Schatz G, Lithgow T (1995) Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria. EMBO J 14 6318-6326... 

Cytosolic Factors Involved in Translocation and Import of Precursors 139... 

The efficient uptake of precursor proteins depends on their presentation in a translocation competent state. This is maintained in vivo by the specific interaction with a highly conserved group of proteins, the heat-shock or stress related proteins (hps70s). These act as molecular chaperones and interact with the proteins to maintain them in a correctly folded state, a process which is ATP dependent. 

Deshaies, R.J., Koch, B.D., Wemer-Washbume, M., Craig, E.A., Schekman, R. (1988). kA subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature 332,800-805. 

In an Escherichia coli expression system for the aqualysin I precursor, the precursor is processed autoproteolytically into the mature 28-kDa enzyme by treatment at 65 ° C.23) In this case, the N-terminal pro-sequence is required for the production of active enzyme and functions to stabilize the precursor structure.283 The C-terminal pro-sequence is not essential for the production of active aqualysin 1,293 but seems to be involved in the translocation of the precursor across the cytoplasmic membrane.303 In a Thermus thermophilus expression system,313 the C-terminal pro-sequence is required for the production and extracellular secretion of active aqualysin I.323 In an E. coli expression system for the subtilisin E gene, the N-terminal pro-sequence is essential for the production of active enzyme,333 as in the case of aqualysin I. The requirement of the pro-sequence is also shown in vitro for the refolding of the inactive mature protein to an active enzyme.34 353 The functions of the N-terminal pro-sequences of aqualysin I and subtilisin E seem to be similar. 

Translocation of Proteins Across Membranes. The transfer of proteins across biological membranes generally involves a hydrolytic modification step of the precursor form of the mature protein. This processing has been shown clearly to occur during segregation of secretory proteins, transport of proteins into mitochondria, and entry of plant and microbial toxins into cells as shown in Table XII. 

Kang, P. J., Ostermann, J., Shilling, )., Neupert, W., Craig, E, A., and Planner, N. (1990). Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature (London) 348, 137-143. 

Throughout this review, terms such as unfolded, loosely folded, or export-competent state are bandied about (no doubt distressing some disciples of protein structure). These terms are not meant to imply a single definable conformation, or the absence of a specific secondary or tertiary structure. In fact, several lines of evidence indicate that a translocation-competent precursor can contain considerable structure. For example, efficient signal peptide function requires that the hydrophobic core assume an a-helical conformation (Jones et ai, 1990), and evidence has been presented that enzymatically active DHFR (Freudl et al., 1988) and a fully folded biotin acceptor domain (Reed and Cronan, 1991)—or conformers of these proteins that are in equilibrium with the native states—can be translocated across the cytoplasmic membrane of E. coli in vivo. 

The processes of prepropolypeptide synthesis, translocation, proteolytic processing and non-proteolytic modification can be enzymatically defined. These definitions are continuing to be developed and clarified. There are limited reports on insect neuropeptide processing (101.102. but these investigations should increase rapidly with the identification of precursor sequences via molecular genetics. The identification of processing enzymes, both proteolytic and non-proteolytic, will further open whole new areas for exploration. 

Fig. 12.5. Biogenesis and assembly of cytochrome 6-c, complex in the inner mitochondrial membrane. Cytochrome fc-Cj complex contains at least five different subunits COREI (corl), COREII (corll), nonheme iron protein (Fe-S), cytochrome c, (cyt Cj), and cytochrome b (cyt b). Cytochrome f> is a mitochondrial gene product and is probably assembled into the inner membrane (IM) via vectorial translation by mitochondrial ribosomes. The other subunits are synthesized on cytoplasmic ribosomes as larger precursors. The precursors, perhaps in association with a cytoplasmic factor , are attached to receptors on the mitochondrial outer membrane (OM). The complex laterally diffuses to the junctions of the outer and inner membranes, and with the help of a hypothetical translocator the precursors are imported across the membrane. Pre-Corl, pre-Corll, and the pre-nonheme iron protein cross the two membranes, whereas cytochrome c, becomes anchored to the outer face of the inner membrane, facing the intermembrane space (IMS). Cytochrome b is assembled inside the inner membrane, and the nonheme iron protein and Corl and Corll are assembled into the matrix side of the inner membrane. The N-terminal extensions are removed by a soluble matrix protease. The N-terminal extension of cytochrome c, is removed in two steps the first is catalyzed by the matrix protease and the second probably by a protease located on the outer face of the inner membrane.

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