617

Among the proteins found in the chloroplast stroma are the enzymes of the Calvin cycle, which function in fixing carbon dioxide during photosynthesis (see Chapter 12). 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 (see Table 13-1).

Experiments with isolated chloroplasts, similar to those with mitochondria illustrated in Figure 13-23, 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 subunits to yield the active rubisco enzyme.

The general process of stromal import appears to be very similar to that of protein import into the mitochondrial matrix (see Figure 13-24). 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.

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.