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 of 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 the A chain passenger protein is resistant to extremely high concentrations of protease, unless deliberately unfolded using denaturant. The A chain moiety is furthermore active, indicating that the presence of the presequence does not prevent formation of a tightly folded, native state. In contrast, receptor-bound p33KRA (fusion protein comprising the 33-kDa presequence plus 22 residues of mature protein, linked to the A chain of ricin) is quantitatively digested by protease concentrations that have little effect on the A chain in solution. We conclude that protein unfolding can take place on the chloroplast surface in the absence of translocation and without the aid of soluble factors.
Walker
D
D
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Chaddock
A M
AM
Chaddock
J A
JA
Roberts
L M
LM
Lord
J M
JM
Robinson
C
C
D002736:Chloroplasts / Q000378:metabolism*; D019286:Endopeptidase K; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012276:Ricin / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*
D010940:Plant Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012276:Ricin; D012697:Serine Endopeptidases; D019286:Endopeptidase K