RICIN-A CHAIN FUSED TO A CHLOROPLAST-TARGETING SIGNAL IS UNFOLDED ON THE CHLOROPLAST SURFACE PRIOR TO IMPORT ACROSS THE ENVELOPE MEMBRANES

The initial stages of chloroplast protein import involve the binding o f precursor proteins to surface-bound receptors prior to translocation across the envelope membranes in a partially folded conformation, We have analyzed the unfolding process by examining the conformation of a construct, comprising the presequence of a chloroplast protein linked to ricin A chain, before and after binding to the chloroplast surface , We show that the presequence is highly susceptible to proteolysis in solution, probably reflecting a lack of tertiary structure, whereas t he A chain passenger protein is resistant to extremely high concentrat ions of protease, unless deliberately unfolded using denaturant, The A chain moiety is furthermore active, indicating that the presence of t he presequence does not prevent formation of a tightly folded, native state, In contrast, receptor-bound p33KRA (fusion protein comprising t he 33-kDa presequence plus 22 residues of mature protein, linked to th e A chain of ricin) is quantitatively digested by protease concentrati ons that have little effect on the A chain in solution, We conclude th at protein unfolding can take place on the chloroplast surface in the absence of translocation and without the aid of soluble factors.