The chloroplast genes ycf3 and ycf4 from the green alga Chlamydomonas reinhardtii have been characterized. The deduced amino acid sequences of Ycf4 (197 residues) and Ycf3 (172 residues) display 41-52% and 64-78% sequence identity, respectively, with their homologues from algae, land plants and cyanobacteria. In C. reinhardtii, ycf4 and ycf3 are co-transcribed as members of the rps9-ycf4-ycf3-rps18 polycistronic transcriptional unit into RNAs of 8.0 kb and 3.0 kb corresponding to the entire unit and to rps9-ycf4-ycf3, respectively. Using biolistic transformation, ycf4 and ycf3 were disrupted with a chloroplast selectable marker cassette. Transformants lacking ycf4 or ycf3 were unable to grow photoautotrophically and were deficient in photosystem I activity. Western blot analysis showed that the photosystem I (PSI) complex does not accumulate stably in thylakoid membranes of these transformants. Ycf4 and Ycf3 were localized on thylakoid membranes but not stably associated with the PSI complex and accumulated to wild-type levels in mutants lacking PSI. RNA blot hybridizations showed that transcripts of psaA, psaB and psaC accumulate normally in these mutants and use of chimeric reporter genes revealed that Ycf3 is not required for initiation of translation of psaA and psaB mRNA. Our results indicate that Ycf3 and Ycf4 are required for stable accumulation of the PSI complex.

The nuclear gene PsaD encodes an abundant chloroplast protein located on the stromal side of the Photosystem I complex. We have cloned and sequenced a genomic fragment containing the PsaD gene from the green alga Chlamydomonas reinhardtii. Sequence comparison with its cDNA revealed that the PsaD ORF contains no introns. Thus, the regulatory sequences required for high-level expression of PsaD must lie in the flanking promoter and untranslated regions. We used this genomic fragment to construct a vector that allows for high-level expression of endogenous and exogenous genes, as well as cDNAs that could not be expressed from existing vectors. It is also possible to use the PsaD transit sequence to target the expressed protein to the chloroplast compartment.

The psbC gene of Chlamydomonas reinhardtii encodes P6, the 43 kd photosystem II core polypeptide. The sequence of P6 is highly homologous to the corresponding protein in higher plants with the exception of the N‐terminal region where the first 12 amino acids are missing. Translation of P6 is initiated at GUG in C. reinhardtii. The chloroplast mutant MA16 produces a highly unstable P6 protein. The mutation in this strain maps near the middle of the psbC gene and consists of a 6 bp duplication that creates a Ser‐Leu repeat at the end of one transmembrane domain. Two nuclear mutants, F34 and F64, and one chloroplast mutant, FuD34, are unable to synthesize P6. All of these mutants accumulate wild‐type levels of psbC mRNA. The FuD34 mutation has been localized near the middle of the 550 bp 5′ untranslated region of psbC where the RNA can be folded into a stem‐loop structure. A chloroplast suppressor of F34 has been isolated that partially restores synthesis of the 43 kd protein. The mutation of this suppressor is near that of FuD34, in the same stem‐loop region. These chloroplast mutations appear to define the target site of a nuclear factor that is involved in P6 translation.

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

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