Oren Ostersetzer

Light-stimulated degradation of an unassembled Rieske FeS protein by a thylakoid-bound protease: the possible role of the FtsH protease.

Unassembled subunits of the cytochrome b6f complex as well as components of other unassembled chloroplastic complexes are rapidly degraded within the organelle. However, the mechanisms involved in these proteolytic processes are obscure. When the Rieske FeS protein (RISP) is imported into isolated chloroplasts in vitro, some of the protein does not property assemble with the cytochrome complex, as determined by its sensitivity to exogenous protease. When assayed in intact, lysed, or fractionated chloroplasts, the imported RISP was found to be sensitive to endogenous proteases as well. The activity responsible for degradation of the unassembled protein was localized to the thylakoid membrane and characterized as a metalloprotease requiring zinc ions for its activity. The degradation rate was stimulated by light, but no involvement of ATP or redox control was observed. Instead, when the RISP that was attached to thylakoid membranes was first illuminated on ice, degradation proceeded in either light or darkness at equal rates suggesting a light-induced conformational change making the protein prone to degradation. Antibodies raised against native FtsH, a bacterial, membrane-bound, ATP-dependent, zinc-stimulated protease, effectively inhibited degradation of the unassembled RISP, suggesting a role for the chloroplastic FtsH in this process.

CRS1, a Chloroplast Group II Intron Splicing Factor, Promotes Intron Folding through Specific Interactions with Two Intron Domains

Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1s specificity for the atpF intron in vivo can be explained by CRS1s intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1s intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments.

The protein disulfide isomerase-like RB60 is partitioned between stroma and thylakoids in Chlamydomonas reinhardtii chloroplasts.

Translation of psbA mRNA inChlamydomonas 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 psbAmRNA. RB60 has been suggested to act as a redox-sensor subunit of the protein complex regulating translation of chloroplastpsbA 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.

ATP-dependent association between subunits of Clp protease in pea chloroplasts.

Abstract. The chloroplast ATP-dependent Clp protease (EC 3.4.21.92) is composed of the proteolytic subunit ClpP and the regulatory ATPase, ClpC. Although both subunits are found in the stroma, the interaction between the two is dynamic. When immunoprecipitation with antibodies against ClpC was performed on stroma from dark-adapted pea (Pisum sativum L. cv. Alaska) chloroplasts, ClpC but not ClpP was precipitated. However, when stroma was supplemented with ATP, both ClpC and ClpP were precipitated. Co-immunoprecipitation was even more efficient in the presence of ATP-γ-S, suggesting that the association between regulatory and proteolytic subunits is dependent on binding of ATP to ClpC, but not its hydrolysis. To further test this association, stroma was fractionated by column chromatography, and the presence of Clp subunits in the different fractions was monitored immunologically. When stroma depleted of ATP was fractionated on an ion-exchange column, ClpP and ClpC migrated separately, whereas in the presence of ATP-γ-S both subunits co-migrated. Similar results were observed in size-exclusion chromatography. To further characterize the precipitated enzyme, its proteolytic activity was assayed by testing its ability to degrade β-casein. No degradation was observed in the absence of ATP, and degradation was inhibited in the presence of phenylmethylsulfonyl fluoride, consistent with Clp being an ATP-dependent serine protease. The activity of the isolated enzyme was further tested using chimeric OE33 as a model substrate. This protein was also degraded in an ATP-dependent manner, supporting the suggested role of Clp protease as a major housekeeping protease in the stroma.

Effects of light and temperature on expression of ClpC, the regulatory subunit of chloroplastic Clp protease, in pea seedlings.

Chloroplasts contain homologues to the proteolytic and regulatory subunits of bacterial ATP-dependent Clp protease. We tested the effects of light and temperature on the expression of ClpC, the chloroplastic homologue of the regulatory subunit. ClpC mRNA was present in all tissues of pea seedlings, most abundantly in leaves. Higher levels of the message were found in green leaves than in etiolated ones. Exposure of etiolated seedlings to light resulted in further accumulation of the transcript. Similarly, ClpC protein level was lower in etiolated leaves, and increased upon exposure to light. Transferring seedlings from 25°C to either 17 or 37°C resulted in a decrease in both ClpC mRNA and protein, with the lower temperature being the most effective.

The Proteolytic Machinery of Chloroplasts: Homologues of Bacterial Proteases

In the past 10–15 years, many examples of non-specific and specific degradation of proteins in the chloroplast have accumulated [for review, see (1)]. These include degradation of photo- and oxidatively damaged proteins, proteins whose levels are regulated by different environmental conditions, unassembled proteins, and proteins lacking their prosthetic groups. However, the proteases involved in their degradation were largely unknown. As a first step toward identification of the proteases involved in these specific proteolytic processes, we identified and characterized several chloroplast proteases, all of which are homologues of bacterial proteases. In this paper, we provide an overview of these proteases, present recent related data, and discuss the implications of these on our understanding of proteolytic processes in chloroplasts.