Chloroplasts are essential organelles in the cells of plants and algae. The functions of these specialized plastids are largely dependent on the ~3000 proteins residing in the organelle. Although chloroplasts are capable of a limited amount of semiautonomous protein synthesis - their genomes encode ~100 proteins - they must import more than 95% of their proteins after synthesis in the cytosol. Imported proteins generally possess an N-terminal extension termed a transit peptide. The importing translocons are made up of two complexes in the outer and inner envelope membranes, the so-called Toc and Tic machineries, respectively. The Toc complex contains two precursor receptors, Toc159 and Toc34, a protein channel, Toc75, and a peripheral component, Toc64/OEP64. The Tic complex consists of as many as eight components, namely Tic22, Tic110, Tic40, Tic20, Tic21 Tic62, Tic55 and Tic32. This general Toc/Tic import pathway, worked out largely in pea chloroplasts, appears to operate in chloroplasts in all green plants, albeit with significant modifications. Sub-complexes of the Toc and Tic machineries are proposed to exist to satisfy different substrate-, tissue-, cell- and developmental requirements. In this review, we summarize our understanding of the functions of Toc and Tic components, comparing these components of the import machinery in green algae through trees. We emphasize recent findings that point to growing complexities of chloroplast protein import process, and use the evolutionary relationships between proteins of different species in an attempt to define the essential core translocon components and those more likely to be responsible for regulation. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

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Heat shock protein 70s (Hsp70s) are encoded by a multigene family and are located in different cellular compartments. They have broad-ranging functions, including involvement in protein trafficking, prevention of protein aggregation, and assistance in protein folding. Hsp70s work together with their cochaperones, J domain proteins and nucleotide exchange factors (e.g., GrpEs), in a functional cycle of substrate binding and release accompanied by ATP hydrolysis. We have taken advantage of the gene targeting capability of the moss Physcomitrella patens to investigate the functions of chloroplast Hsp70s. We identified four Hsp70 genes and two GrpE cochaperone homolog genes (CGE) in moss that encode chloroplast proteins. Disruption of one of the Hsp70 genes, that for Hsp70-2, caused lethality, and protein import into heat-shocked chloroplasts isolated from temperature-sensitive hsp70-2 mutants was appreciably impaired. Whereas the double cge null mutant was not viable, we recovered a cge1 null/cge2 knock down mutant in which Hsp70-2 was upregulated. Chloroplasts isolated from this mutant demonstrated a defect in protein import. In addition, two different precursors staged as early import intermediates could be immunoprecipitated with an Hsp70-2-specific antibody. This immunoprecipitate also contained Hsp93 and Tic40, indicating that it represents a precursor still in the Toc/Tic translocon. Together, these data indicate that a stromal Hsp70 system plays a crucial role in protein import into chloroplasts.

Crd1 (Copper response defect 1), which is required for the maintenance of photosystem I and its associated light-harvesting complexes in copper-deficient (-Cu) and oxygen-deficient (-O(2)) Chlamydomonas reinhardtii cells, is localized to the thylakoid membrane. A related protein, Cth1 (Copper target homolog 1), is shown to have a similar but not identical function by genetic suppressor analysis of gain-of-function sct1 (suppressor of copper target 1) strains that are transposon-containing alleles at CTH1. The pattern of Crd1 versus Cth1 accumulation is reciprocal; Crd1 abundance is increased in -Cu or -O(2) cells, whereas Cth1 accumulates in copper-sufficient (+Cu), oxygenated cells. This expression pattern is determined by a single trans-acting regulatory locus, CRR1 (COPPER RESPONSE REGULATOR 1), which activates transcription in -Cu cells. In +Cu cells, a 2.1-kb Cth1 mRNA is produced and translated, whereas Crd1 is transcribed only at basal levels, leading to Cth1 accumulation in +Cu cells. In -Cu cells, CRR1 function determines the activation of Crd1 expression and the production of an alternative 3.1-kb Cth1 mRNA that is extended at the 5' end relative to the 2.1-kb mRNA. Synthesis of the 3.1-kb mRNA, which encodes six small upstream open reading frames that possibly result in poor translation, blocks the downstream promoter through transcriptional occlusion. Fluorescence analysis of wild-type, crd1, and sct1 strains indicates that copper-responsive adjustment of the Cth1:Crd1 ratio results in modification of the interactions between photosystem I and associated light-harvesting complexes. The tightly coordinated CRR1-dependent regulation of isoenzymes Cth1 and Crd1 reinforces the notion that copper plays a specific role in the maintenance of chlorophyll proteins.

Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the ApH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a proteaseresistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts.