Abstract
Translation of psbA mRNA in Chlamydomonas reinhardtii chloroplasts is regulated by a redox signal(s). RB60 is a member of a protein complex that binds with high affinity to the 5'-untranslated region of psbA mRNA. RB60 has been suggested to act as a redox-sensor subunit of the protein complex regulating translation of chloroplast psbA mRNA. Surprisingly, cloning of RB60 identified high homology to the endoplasmic reticulum-localized protein disulfide isomerase, including an endoplasmic reticulum-retention signal at its carboxyl terminus. Here we show, by in vitro import studies, that the recombinant RB60 is imported into isolated chloroplasts of C. reinhardtii and pea in a transit peptide-dependent manner. Subfractionation of C. reinhardtii chloroplasts revealed that the native RB60 is partitioned between the stroma and the thylakoids. The nature of association of native RB60, and imported recombinant RB60, with thylakoids is similar and suggests that RB60 is tightly bound to thylakoids. The targeting characteristics of RB60 and the potential implications of the association of RB60 with thylakoids are discussed.
Highlights
The chloroplast contains a small circular genome that encodes about 5–10% of the chloroplast proteins [1]
RRB60 Is Imported into C. reinhardtii Chloroplasts—The targeting of proteins synthesized by cytoplasmic ribosomes to the chloroplast is typically determined by an amino-terminal transit peptide
RB60 was first isolated as a component of a protein complex which assembles with high affinity on the 5Ј-untranslated region (5Ј-UTR) of C. reinhardtii chloroplast psbA mRNA [19]
Summary
The chloroplast contains a small circular genome that encodes about 5–10% of the chloroplast proteins [1]. RRB60 Is Imported into C. reinhardtii Chloroplasts—The targeting of proteins synthesized by cytoplasmic ribosomes to the chloroplast is typically determined by an amino-terminal transit peptide. To test whether the amino-terminal sequence of rRB60 could direct import into chloroplasts, radiolabeled rRB60 was synthesized in vitro and incubated with C. reinhardtii chloroplasts isolated by Percoll step gradient.
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