Protein Import and Routing Systems of Chloroplasts

Abstract

Plastids are ubiquitous plant cell organelles that perform many essential functions. Chloroplasts are the most complex type of plastid, both structurally and functionally, containing six distinct compartments and performing essential processes, such as fatty acid and amino acid biosynthesis in addition to their well-known role in photosynthesis. The many plastid proteins needed for these and other essential metabolic pathways derive from two genetic systems. The plastid genome encodes ∼100 proteins (Sugiura, 1989), but the vast majority of plastid proteins are encoded by nuclear genes and synthesized as precursors in the cytoplasm. Consequently, specific and reliable protein transport mechanisms are needed to direct these precursor proteins to their proper location within chloroplasts. Most studies of protein transport to and within chloroplasts have been performed with isolated organelles, whereby the general process of protein transport, as schematically presented in Figure 1, can be broken down into three distinct phases: specific targeting of precursor proteins to plastids, transport across the two envelope membranes, and in many cases, sorting to the proper plastidic compartment. One major conclusion derived from these studies is that proper localization of cytoplasmically synthesized chloroplastic proteins is accomplished by specific interactions between targeting sequences contained within the precursor proteins and the transport machinery contained within chloroplasts. This review focuses on the transport process and the machinery that mediates it; it also includes a brief consideration of targeting sequences. Many excellent reviews have covered earlier progress in understanding chloroplastic protein transport (Keegstra et al., 1989; de Boer and Weisbeek, 1991; Schnell, 1995, 1998; Cline and Henry, 1996; Kouranov and Schnell, 1996; Fuks and Schnell, 1997; Lübeck et al., 1997a; Heins et al., 1998); these reviews can be consulted for additional details. Most precursor proteins are larger than their corresponding plastid-localized forms. The N-terminal transit peptide that is cleaved off the precursor protein upon entry into chloroplasts (Figure 1) usually contains stromal-targeting information that is necessary and sufficient for transport of precursors across the two envelope membranes (Keegstra et al., 1989; de Boer and Weisbeek, 1991). In some cases, the cleavable transit peptide is bipartite and contains additional targeting information, as is best exemplified by proteins destined for the thylakoid lumen. In addition to the stromal-targeting domain, bipartite peptides of lumenal proteins contain a second domain that directs transport across the thylakoid membrane (Figure 1; de Boer and Weisbeek, 1991; Cline and Henry, 1996). The two domains of these bipartite transit peptides are removed by two separate processing proteases (Figure 1, path 3): one in the stroma (VanderVere et al., 1995), and one in the thylakoid lumen (Chaal et al., 1998). Many proteins, especially those destined for the inner envelope membrane or the thylakoid membrane, have both a cleavable stromal-targeting domain and additional targeting information retained within the mature protein (Figure 1, paths 2 and 4). A few precursors, especially those destined for the outer envelope membrane, do not have cleavable targeting sequences (Figure 1, path 6). In the latter case, information directing the protein to its proper location is contained within the mature protein (Cline and Henry, 1996). Despite their common function, stromal-targeting domains from various precursor proteins may vary in length from 30 to >100 amino acids. Interestingly, they share minimal sequence similarity so that no consensus targeting sequences have been established, and efforts to identify common secondary structures have not been successful (Keegstra et al., 1989; von Heijne et al., 1989; von Heijne and Nishikawa, 1991). Most stromal-targeting domains are rich in serine and threonine but deficient in acidic amino acids (Keegstra et al., 1989). The lumenal-targeting domain from bipartite transit peptides has strong similarity to the hydrophobic signal sequences found in secreted proteins (Cline and Henry, 1996). The targeting information contained within mature proteins generally is located in membrane-spanning domains, where it also can serve as a membrane insertion or a stop-transfer signal (Cline and Henry, 1996). Schematic Representation of Pathways Responsible for Targeting Proteins to Their Proper Location within Chloroplasts. The large majority of precursor proteins have a cleavable transit peptide (shown in red). In most cases, the transit peptide includes a stromal-targeting domain that initiates transport of the precursor through the general import pathway (shown in gray). The driving force for protein translocation is thought to be provided by molecular chaperones (MC) that pull precursor proteins into plastids. Precursors lacking additional targe