(Received for publication, October 23, 1995; and in revised form, November 28, 1995)
From the
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.
The translocation of proteins across the chloroplast envelope is a complex process requiring the activities of a number of proteins in both the outer and inner envelope membranes (reviewed in (1) ). Imported proteins are initially synthesized with amino-terminal presequences which contain specific targeting signals that ensure recognition by receptors on the chloroplast surface and subsequent translocation into the stroma, probably at contact sites between the two membranes(2) . The molecular nature of the chloroplast-targeting signals have remained elusive, but considerable progress has been made in identifying and characterizing components of the import apparatus in the envelope membranes(3, 4, 5, 6) .
Some aspects of the overall import mechanism have been largely elucidated, for example requirements for ATP hydrolysis in both the binding and translocation processes(7, 8) , but other aspects remain unclear. Of particular interest are the conformations of proteins during the early stages of the import process. Import intermediates have been identified which span both membranes, demonstrating that precursor proteins traverse the envelope in an unfolded or partially folded conformation(2, 9) . Because chloroplast protein import can proceed entirely posttranslationally, the key question arises as to how and when this unfolding is accomplished and in particular whether it is achieved before or after the precursor interacts with the chloroplast. Perhaps surprisingly, rather few studies have examined the conformations of precursor proteins in solution, possibly because most studies have employed in vitro translation systems in which proteins are synthesized in minute quantities, precluding detailed structural studies on the precursor proteins at any stage of the import process. There is some evidence that chloroplast protein presequences are substantially, if not completely unfolded(10, 11) , but there is no evidence for the presence of a factor in cell-free translation systems which maintains chloroplast precursor proteins in an unfolded conformation. On the contrary, the precursor of 5-enolpyruvylshikimate-3-phosphate 1-carboxyvinyltransferase was found to be catalytically active, and thus presumably correctly folded, after synthesis in a reticulocyte lysate(12) , and protease sensitivity studies on the precursor of a 23-kDa thylakoid lumen protein suggested that this protein comprised an unfolded presequence together with a folded mature moiety(13) . However, the only precursor protein to be studied in detail has been that of ferredoxin(10) , and this study concluded that the entire precursor protein was relatively unfolded. This may be because folding of this particular protein requires formation of the FeS groups, but it nevertheless remains the case that the structures of chloroplast protein precursors in general are poorly understood.
The
driving force for the unfolding process is equally poorly defined and
only one study has addressed the conformation of a protein following
binding to the chloroplast. In this study (14) it was found
that a presequence-dihydrofolate reductase construct was highly
sensitive to proteolysis once bound to the chloroplast, whereas
proteases converted the precursor protein primarily to mature size
dihydrofolate reductase in solution. The interpretation was that the
protein was relatively unfolded once bound to receptors. Furthermore,
additional studies suggested that the unfolding activity may be quite
powerful: dihydrofolate reductase becomes even more tightly folded once
a substrate analog, methotrexate, binds in the active site, to the
extent that translocation of chimeras into mitochondria is blocked (15) . However, this treatment did not prevent import into
chloroplasts (16) , raising the possibility that a particularly
potent unfolding activity is associated with the surface-exposed import
machinery. In this report we have investigated the import
characteristics of a different fusion protein (p33KRA) ()in
which the presequence of a 33-kDa thylakoid lumen protein (33K) is
linked to ricin A chain. Previous work (17) has shown this
protein to be efficiently imported into intact chloroplasts, and assays
are available to assess whether this protein is active even as an in vitro translation product. We show that the fusion protein
is highly active in solution and that the A chain is remarkably
resistant to proteolysis as a result of its tightly folded native
conformation. We also show that the protein becomes susceptible to
digestion once bound to the chloroplast import machinery, providing
evidence that the chloroplast is able to partially unfold tightly
folded proteins without the assistance of soluble import factors.
Figure 1: Proteinase K converts p33KRA predominantly to mature size A chain. Left-hand panel, p33KRA was synthesized in a wheat germ lysate and incubated with proteinase K at the concentrations indicated above the lanes for 40 min at 0 °C. After incubation, samples were analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. RA denotes mobility of authentic mature size A chain marker. Right-hand panel, p33KRA was incubated in the absence of protease or the presence of 50 µg/ml proteinase K as indicated; the sample denoted imp represents the results of importing p33KRA into intact pea chloroplasts. 33KRA represents imported p33KRA which has been proteolytically processed in the chloroplasts to yield A chain containing 22 residues of mature 33K at the amino terminus.
Further indications of the folding characteristics of A chain are shown in Fig. 2. In this experiment we tested whether A chain becomes more susceptible to proteolysis when deliberately unfolded in urea (to exclude the unlikely possibility that the A chain sequence does not contain proteinase K cleavage sites). p33KRA was incubated with 8 M urea to induce unfolding, and the mixture was then diluted 4-fold with buffer containing proteinase K. The data show that this treatment renders the A chain far more susceptible to digestion when compared with the minus-urea control, with the majority of p33KRA now being cleaved to low molecular weight forms (defined here as polypeptides smaller than mature size A chain) by concentrations of proteinase K of 50 µg/ml or greater. Quantitation of the data (shown as part of Fig. 4) indicates that a small proportion of p33KRA continues to be converted to mature size A chain, even at very high protease concentrations; possibly, these are molecules that were able to quickly refold into their correct tertiary structures when diluted from 8 to 2 M urea, whereas the majority were too slow and were degraded. Overall, however, the data provide compelling evidence that ricin A chain is highly resistant to proteolysis as a consequence of its tightly folded three-dimensional structure.
Figure 2: 33KRA is protease-sensitive when deliberately unfolded in urea. p33KRA was synthesized as detailed in the legend to Fig. 1and then diluted 4-fold into 50 mM Hepes, pH 8.0, 330 mM sorbitol-containing proteinase K (PK) at the indicated concentrations in µg/ml (left-hand panel) or incubated with 8 M urea for 30 min on ice, then diluted 4-fold with the same buffer containing proteinase K (right-hand panel). After incubation for 40 min, samples were analyzed as in Fig. 1.
Figure 4: Receptor-bound p33KRA is protease-sensitive and hence probably unfolded. Autoradiogram (A): p33KRA was incubated with pea chloroplasts in the presence of 10 µM MgATP as detailed under ``Experimental Procedures,'' and the chloroplasts were subsequently washed to remove unbound molecules. Aliquots of the chloroplasts were then incubated with proteinase K (PK) at the indicated concentrations (in µg/ml), and one aliquot was incubated with 1 mM MgATP to chase the bound p33KRA into the organelles (lane Ch). Symbols are the same as in Fig. 1and Fig. 2. The graph (B) shows a quantitative assessment of the percentage degradation of p33KRA to low molecular mass polypeptides (defined as smaller than mature size A chain) in the experiment outlined above (=bound graph) or when soluble p33KRA is incubated with proteinase K in the absence or presence of urea as shown in Fig. 2(graphs -/+ urea).
Figure 3: p33KRA has ribosomal RNA N-glycosidase activity. p33KRA (incubation 1) and A chain (incubation 2) were synthesized in a reticulocyte lysate for 60 min prior to extraction of rRNA as detailed(18) . A translation reaction was also conducted following transcription of pGem4Z vector containing no insert (incubation 3). Samples of the rRNA, and of rRNA that was extracted without prior incubation with transcription mixture (incubation 4), were blotted onto nitrocellulose before(-) or after (+) incubation with aniline. Release of a 390-base fragment (390 b) was detected by Northern blotting as detailed under ``Experimental Procedures.''
In summary, we have provided further evidence that proteins can be targeted into chloroplasts without the benefit of a cytosolic antifolding factor and that the unfolding process instead takes place once bound to the chloroplasts, but before translocation across the envelope membranes. The identity of the ``unfoldase'' remains to be established. The precursor proteins may be passed on to an unfoldase activity after interaction with the initial import receptor, or the import receptor may fulfil both roles. It is also possible that envelope lipids play a role in the unfolding process, although it seems unlikely that lipids could achieve this level of unfolding without the input of dedicated proteins. Finally, it is interesting to speculate that the ATP hydrolysis required for stable binding of precursors may well be used to drive the unfolding process, possibly by Hsp70 molecules, which are known to be present in the import complex(9) . This could be achieved either by an ``active'' unfoldase activity in which the protein involved physically unfolds the precursor protein or a ``passive'' unfolding in which the precursor protein ``breathes'' and partially folded forms are progressively stabilized by the unfoldase. Clearly, this topic merits further attention in order to unravel in detail the early events in chloroplast import and in understanding the factors responsible for the presumed unfolding of ricin A chain within the endoplasmic reticulum of mammalian cells.