Translocation competence

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. 

A growing body of evidence indicates that in addition to a targeting element, membrane translocation also requires that the secretory protein assumes a loosely folded or translocation-competent state. The focus of this review is the role that SecB and other molecular chaperones play in sponsoring efficient protein secretion in E. coli. 

Loss of translocation competence could result from burial of the signal peptide within the folded precursor, but at least in the case of preMBP, the signal peptide is still accessible as determined by its selective sensitivity to proteolysis and its ability to bind amphiphiles (Dierstein and Wickner, 1985). In the case of the methotrexate-stabilized COX/DHFR fusion, the COX target peptide is clearly accessible since the precursor binds to energized mitochondria (a target peptide-dependent reaction), and the target peptide is susceptible to proteolysis by partially purified matrix processing enzyme (Eilers and Schatz, 1986). 

B. Correlation between Precursor Folding and Loss of Translocation Competence... 

Consistent with an antifolding role, 25% of the preMBP trapped within secB null strains by the addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) folds into a protease-resistant conformation, while in secB cells, only 5% folds (Kumamoto and Gannon, 1988). Given that protease resistance is a less sensitive probe of preMBP conformation than export competence [in vitro the translocation competence of preMBP decays more rapidly than its protease sensitivity (Weiss et al., 1989)], then this protease-resistant fraction and the fraction of preMBP that becomes export incompetent in secB null strains are quantitatively similar (25% vs. 40%, respectively). Hence the fraction of MBP that is unable to be translocated in the absence of SecB represents the folded fraction. 

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. 

Once the synthetic machinery/nascent protein complex has been delivered to the membrane by SRP, it interacts with the membrane to form a translocation-competent species. It is in this process that we envision the signal sequence to play its most active and crucial part. In Section VIII we propose a model for the initial interactions of a signal sequence with the membrane. These interactions bring the signal sequence and nascent protein into the proper orientation for membrane binding and effect the initial entry of the protein into the membrane interior, readying it for the next step, which is translocation. 

Proproteins interact with molecular chaperones in loosely-folded, translocation-competent conformations, particularly for a polypeptide chain targeted for the post-translalional process. 

The precursor proteins are maintained in a translocation-competent state by the folding-antagonizing activity of their signal peptides and cytoplasmic chaperones, which potentially include the signal recognition particle (SRP) and the FtsY protein [47-49]. This enables translocation across the membrane through the Sec apparatus, which can only handle unfolded or loosely folded proteins. 

Chemical energy in the form of ATP hydrolysis is required to keep the protein in the translocation competent state and possibly also during the translocation step. Import into mitochondria also requires an electrical potential across the inner mitochondrial membrane in contrast, chloroplast import has no such requirement . 

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