INTRODUCTION . SYNTHESIS . Magnitude of the Problem .. In Vitro Chloroplast Protein Synthesis .. Systems . Products .. Are a/l chloroplast-synthesized polypeptides encoded in chloroplast DNA? . Are Sorghum mesophyf/ chloroplasts diJforent? . In Vivo Systems . Use of inhibitors . Evidence from heat-treated plants . Evidence from mutants .. Does the Site of Synthesis Matter? .. TRANSPORT . ASSEMBLY . CONTROL . RuBP Carboxylase . Peak D . Chlorophyl l alb Binding Complex . Do Ch70roplasts Export Regulatory Products 0/ Transcription or Translation? ..... . THE OVERALL PICTURE .

Chloroplast Proteins: Synthesis, Transport, and Assembly

Chimaeric genes can be constructed which fuse the transit peptide of a small subunit of the chloroplast-located ribulose 1,5-bisphosphate carboxylase with a bacterial protein. The fusion protein is translocated into chloroplasts and cleaved in a similar way to the small subunit polypeptide precursor.

Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase.

The role of the transit peptide in the routing of precursors toward different chloroplast compartments

Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts.

Changes in light energy distribution upon state transitions: an in vivo photoacoustic study of the wild type and photosynthesis mutants from Chlamydomonas reinhardtii

Genetic analysis of 25 nuclear mutants defective in the chlorophyll-protein complex CP1 was undertaken. The mutants belong to 13 complementation groups scattered throughout the nuclear genome. All these mutants lack the apoprotein of CP1 and, in addition, a specific set of six low molecular weight thylakoid polypeptides. System I particles obtained by treating WT thylakoid membranes with detergent specifically contain those polypeptides which the mutants lack. These observations suggest that a particular sub-structure of the thylakoid membrane associated with the photosystem I activity is missing from all 25 mutants studied, and that this general phenotype can result from mutation at any one of several unlinked Mendelian loci.

Studies on mutants deficient in the photosystem I reaction centers in Chlamydomonas reinhardtii.

The small subunit (S) of ribulose-1.5-bisphosphate carboxylase (RuBPCase) has provided a model system to probe the sequence,of events surrounding the synthesis and transport of cytoplasmically-synthesized proteins into chloroplasts.l-’ S is synthesized initially on free cytoplasmic poly~omes~.’.~ as a precursor (pS) which is 4,000 to 5,000 daltons larger than the mature polypeptide.’-’ After translation in a wheat germ cell-free system, the precursor can be transported into purified, intact chloroplasts, processed to the mature form,*-‘ and the latter assembled with endogenous large subunits in the chloroplast stroma to form the holoenzyme.’~’ The posttranslational nature of pS transport indicates that the mechanism must be necessarily different from that used for the cotranslational segregation of secretory proteins into the cisternae of rough endoplasmic reticulum.’ Because pS is a major translation product synthesized from plant poly A RNA in a wheat germ cell-free system,’,’,’ the post-translational transport and processing of pS could be detected easily in spite of suboptimal conditions for reconstitution of protein transport in vitro. However, to determine how less abundant, cytoplasmically synthesized proteins are imported into chloroplasts it is necessary to develop a cell-free reconstitution system with high efficiency and sensitivity. Therefore, we have examined quantitatively the conditions that lead to improved polypeptide uptake into chloroplasts in vitro. Under these conditions, a large number of pea polypeptides synthesized in the wheat germ cell-free system are transported posttranslationally through the chloroplast envelope and become localized in the stroma or associated with the thylakoid membranes.

Post-translational uptake of cytoplasmically synthesized proteins by intact chloroplasts in vitro*

Recent studies have established that transport across chloroplast envelopes of proteins which are synthesized by cytoplasmic ribosomes can occur by a post-translational mechanism (1,2). Dobberstein et al. (3) first discovered that a major chloroplast stromal protein, the small subunit (S) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) is synthesized by free polysomes in the green alga, Chlamydomonas reinhardtii. Moreover, they found that translation of the small subunit messenger RNA in vitro yields a precursor (pS) 4000–5000 daltons larger than the mature protein. Upon incubation with a cell-free Chlamydomonas extract pS can be processed to the mature form and a small peptide fragment designated the transit peptide (4,5). Dobberstein et al. (3) proposed that transport of the RuBPCase small subunit in vivo occurs after it is completely synthesized and that the transit sequence on pS facilitates its post-translational interaction with the chloroplast envelope. This proposed mechanism is fundamentally distinct from the co-translational transport across endoplasmic reticulum membranes of proteins which are synthesized by membrane-bound ribosomes (6,7). Precursor forms of the RuBPCase small subunit also have been found among the translation products of spinach (1), pea (1,2,8) and duckweed (9) mRNA in cell-free systems.

In Vitro Synthesis, Transport, and Assembly of the Constituent Polypeptides of the Light-Harvesting Chlorophyll a/b Protein Complex

It is well established that the chloroplast synthesizes only a limited number of its own proteins. Many stromal enzymes and thyla-koid membrane polypeptides are made in the cytosol and must be transferred subsequently from their sites of synthesis to their final locations inside the chloroplast (1). The mechanism of intracellular transport of proteins across the two chloroplast envelope membranes and the events associated with it are unknown.

In Vitro Synthesis, Transport, and Assembly of Ribulose 1, 5-Bisphosphate Carboxylase Subunits

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Studies on mutants deficient in the photosystem I reaction centers in Chlamydomonas reinhardtii.

Post-translational uptake of cytoplasmically synthesized proteins by intact chloroplasts in vitro*

In Vitro Synthesis, Transport, and Assembly of the Constituent Polypeptides of the Light-Harvesting Chlorophyll a/b Protein Complex

In Vitro Synthesis, Transport, and Assembly of Ribulose 1, 5-Bisphosphate Carboxylase Subunits