We have purified a nascent-polypeptide-associated complex (NAC) which prevents short ribosome-associated nascent polypeptides from inappropriate interactions with proteins in the cytosol. NAC binds nascent-polypeptide domains emerging from ribosomes unless a signal peptide is fully exposed. Depletion of cytosolic proteins (including NAC) from ribosomes carrying nascent polypeptides allows the signal recognition particle (SRP) to crosslink to polypeptides irrespective of whether or not they contain signal peptides. In the absence of cytosol, proteins lacking signal peptides can be mistranslocated into the endoplasmic reticulum in vitro, albeit with low efficiency. Readdition of NAC restores the specificity of SRP and fidelity of translocation.

Recent evidence supports the view that cellular protein folding may be mediated by molecular chaperones. A fundamental question concerns the stage in its biogenesis at which the folding protein makes frst contact with these components. We show here by crosslinking that the chaperone DnaJ binds nascent ribosome-bound polypeptide chains as short as 55 residues. Cotranslational binding of DnaJ to firefly luciferase and chloramphenicol acetyltransferase resulted in an arrest of folding as long as the functional partners of DnaJ in Escherichia col, DnaK and GrpE, were missing. Protein uptake into microsomes and mitochondria was also interrupted by DnaJ. Both folding and post-transltional translocation recommenced upon addition of DnaK and GrpE. We propose that DnaJ protects nascent polypeptide chains against aggregation and, in cooperation with Hsp7O, controls their productive folding once a complete polypeptide or a polypeptide domain has been synthesized.

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