Among the proteins found in the chloroplast stroma are the enzymes of the Calvin cycle, which function in fixing car bon dioxide during photosynthesis. The large (L) subunit of ribulose 1,5-bisphosphate carboxylase (rubisco) is encoded by chloroplast DNA and synthesized on chloroplast ribosomes in the stromal space. The small (S) subunit of rubisco and all the other Calvin cycle enzymes are encoded by nuclear genes and transported to chloroplasts after their synthesis in the cytosol. The precursor forms of these stromal proteins contain an N-terminal stromal-import sequence.
Experiments with isolated chloroplasts, similar to those with mitochondria illustrated in Figure 1, have shown that they can import the rubisco S-subunit precursor after its synthesis. After the unfolded precursor enters the stromal space, it binds transiently to a stromal Hsp70 chaperone, and the N-terminal stromal-import sequence is cleaved. In reactions facilitated by Hsp60 chaperonins that reside within the stromal space, eight S subunits combine with eight L sub units to yield the active rubisco enzyme.
Fig1. Import of mitochondrial precursor proteins is assayed in a cell-free system. Mitochondrial precursor proteins with attached uptake-targeting signals can be synthesized on ribosomes in a cell-free reaction. When respiring mitochondria are added to these synthesized mitochondrial precursor proteins (top), the proteins are taken up by mitochondria. Inside mitochondria, the proteins are protected from the action of proteases such as trypsin. When no mitochondria are present (bottom), the mitochondrial proteins are degraded by added protease. Only energized (respiring) mitochondria, which have a proton electrochemical gradient (proton-motive force) across the inner membrane, take up these proteins. Furthermore, the imported proteins must contain an appropriate uptake-targeting sequence. Uptake also requires ATP and a cytosolic extract containing chaperone proteins that maintain the precursor proteins in an unfolded conformation. This assay has been used to study targeting sequences and other features of the translocation process.
The general process of stromal import appears to be very similar to that of protein import into the mitochondrial matrix (see Figure 2). At least three chloroplast outer-membrane proteins, including a receptor that binds the stromal-import sequence and a translocation channel protein, and five chloroplast inner-membrane proteins are known to be essential for directing proteins to the stroma. Although these proteins are functionally analogous to the receptor and channel proteins in the mitochondrial membrane, they are not structurally homologous. The lack of sequence similarity between these chloroplast and mitochondrial proteins suggests that they may have arisen independently during evolution.
Fig2. Protein import into the mitochondrial matrix. Precursor proteins synthesized on cytosolic ribosomes are main tained in an unfolded or partially folded state by bound chaperones, such as cytosolic Hsp70 (step 1). After a precursor protein binds to an import receptor near a site of contact with the inner membrane (step 2), it is transferred into the general import pore (step 3). The translocating protein then moves through this channel and an adjacent channel in the inner membrane (steps 4–5). Note that translocation occurs at rare “contact sites” at which the inner and outer membranes appear to touch. Binding of the translocating protein by matrix Hsp70 and subsequent ATP hydrolysis by Hsp70 helps drive import into the matrix. Once the targeting sequence is removed by a matrix protease and Hsp70 is released from the newly imported protein (step 6), the protein folds into its mature, active conformation within the matrix (step 7). Folding of some proteins depends on matrix chaperonins. See G. Schatz, 1996, J. Biol. Chem. 271:31763, and N. Pfanner et al., 1997, Annu. Rev. Cell Dev. Biol. 13:25.
The available evidence suggests that chloroplast stromal proteins, like mitochondrial matrix proteins, are imported in the unfolded state. Import into the stroma depends on ATP hydrolysis catalyzed by a stromal Hsp70 chaperone whose function is similar to that of Hsp70 in the mitochondrial matrix and BiP in the ER lumen. Unlike mitochondria, chloroplasts do not generate an electrochemical gradient (proton-motive force) across their inner membrane. Thus protein import into the chloroplast stroma appears to be powered solely by ATP hydrolysis.
