Chloroplast protein targeting involves localized translation in Chlamydomonas

Abstract

The compartmentalization of eukaryotic cells requires that newly synthesized proteins be targeted to the compartments in which they function. In chloroplasts, a few thousand proteins function in photosynthesis, expression of the chloroplast genome, and other processes. Most chloroplast proteins are synthesized in the cytoplasm, imported, and then targeted to a specific chloroplast compartment. The remainder are encoded by the chloroplast genome, synthesized within the organelle, and targeted by mechanisms that are only beginning to be elucidated. We used fluorescence confocal microscopy to explore the targeting mechanisms used by several chloroplast proteins in the green alga Chlamydomonas. These include the small subunit of ribulose bisphosphate carboxylase (rubisco) and the light-harvesting complex II (LHCII) subunits, which are imported from the cytoplasm, and 2 proteins synthesized in the chloroplast: the D1 subunit of photosystem II and the rubisco large subunit. We determined whether the targeting of each protein involves localized translation of the mRNA that encodes it. When this was the case, we explored whether the targeting sequence was in the nascent polypeptide or in the mRNA, based on whether the localization was translation-dependent or -independent, respectively. The results reveal 2 novel examples of targeting by localized translation, in LHCII subunit import and the targeting of the rubisco large subunit to the pyrenoid. They also demonstrate examples of each of the three known mechanisms-posttranslational, cotranslational (signal recognition particle-mediated), and mRNA-based-in the targeting of specific chloroplast proteins. Our findings can help guide the exploration of these pathways at the biochemical level.

Fig. 1.

Translation-dependent localization of the chloroplast rbcL mRNA at the pyrenoid for LSU targeting. (A) An illustration of the cell shown in (C) demonstrating the single chloroplast (green) with its lobes and globular basal region. The basal region contains the pyrenoid (P; blue). Also indicated are the cytosolic region (gray) and the approximate locations of T zones (T), the nucleus (N), and the flagella. The region in which rbcL mRNAs localize is shown in red. (B) An ML cell showing the rbcL mRNA signal and immunolabeled rubisco. (C–F) Signals from the rbcL and psbA mRNAs in cells from constant ML (C), DA (D), ML with lincomycin (E), and HL (F) for 60 min. The punctate colocalized psbA and rbcL mRNA signals near the pyrenoid in (E) and (F) are chloroplast stress granules, which form in response to oxidative stress caused by a secondary effect of lincomycin (photosensitization to ML) or HL, respectively (21). The micrographs show 0.2-μm optical sections. For each experiment, n ≥ 20 cells. Each pattern was observed in ≥ 80% of cells. (Scale bars: 1 μm.)

Fig. 2.

Translation-independent localization of the psbA mRNA in T zones for de novo PSII assembly. (A–C) Fluorescence signals from the psbA mRNA and the chloroplast ribosomal protein L2 in cells from the following conditions: 2-h DA (A), after a 5-min ML exposure to initiate psbA translation for de novo PSII assembly (ML5′) (B), and ML5′ cells generated in the presence of lincomycin (C). The fourth image column shows the merged channels, with the strongest colocalized signals highlighted in white. The punctate psbA mRNA signal near the pyrenoid that does not colocalize with L2 is in chloroplast stress granules (21). The micrographs show 0.2-μm optical sections. (Scale bar: 1 μm.) (D) The percentage of pixels in sampled T zones with strong colocalized signals [white pixels in (A–C)] for each of the 3 conditions. The error bars indicate 2 standard errors. For each experiment, n ≥ 20 cells.

Fig. 3.

Localization of the psbA mRNA for the de novo assembly and repair of PSII. (A–E) Fluorescence signals from the psbA mRNA and the chloroplast ribosomal protein S-21 in cells from the following conditions: a 2-h DA (A); DA cells exposed to HL for 1 min (HL1′) to induce psbA translation for de novo PSII assembly (B); HL1′ cells generated in the presence of lincomycin (C); HL1′ cells exposed to ML for 5 min, a condition of PSII repair (HL1′ML5′) (D); and HL1′ML5′ cells generated in the presence of lincomycin (E). The micrographs show 0.2-μm optical sections. (Scale bar: 1 μm.) (F) The percentages of pixels with strong colocalized signals in T zones (white bars) and chloroplast lobes (shaded bars) across all cells from the 5 conditions. The error bars indicate 2 standard errors. For each experiment, n ≥ 20 cells.

Fig. 4.

FISH analyses of 2 nucleocytosolic mRNAs. (A) An ML cell showing the distribution of the LhcII mRNAs relative to the chloroplast, stained by immunolabeling L2. The closed-head arrows indicate colocalization near the chloroplast basal region. (B) An ML cell showing the fluorescence signals from LhcII mRNAs and the cytoplasmic ribosomal protein cyL4. (C) An ML cell exposed to puromycin for 1 min that was FISH-probed for the LhcII mRNAs and immunostained for cyL4. (D) An ML cell showing the distribution of the RbcS2 mRNAs relative to the chloroplast, which was FISH-probed for the psbC mRNA. (E) An ML cell that was FISH-probed for the RbcS2 mRNA and immunostained for the cytoplasmic ribosomal protein cyL4. The open-head arrow shows the autofluorescence resulting from excitation at 633 nm, seen in the nonprobed/immunostained cell in (F). (G) An ML cell that was FISH-probed for LhcII and RbcS2 mRNAs. DIC, differential interference contrast. The micrographs show 0.2-μm optical sections. (Scale bar: 1 μm.)