Photosystem II Assembly and Repair Are Differentially Localized inChlamydomonas

Abstract

Many proteins of the photosynthesis complexes are encoded by the genome of the chloroplast and synthesized by bacteriumlike ribosomes within this organelle. To determine where proteins are synthesized for the de novo assembly and repair of photosystem II (PSII) in the chloroplast of Chlamydomonas reinhardtii, we used fluorescence in situ hybridization, immunofluorescence staining, and confocal microscopy. These locations were defined as having colocalized chloroplast mRNAs encoding PSII subunits and proteins of the chloroplast translation machinery specifically under conditions of PSII subunit synthesis. The results revealed that the synthesis of the D1 subunit for the repair of photodamaged PSII complexes occurs in regions of the chloroplast with thylakoids, consistent with the current model. However, for de novo PSII assembly, PSII subunit synthesis was detected in discrete regions near the pyrenoid, termed T zones (for translation zones). In two PSII assembly mutants, unassembled D1 subunits and incompletely assembled PSII complexes localized around the pyrenoid, where we propose that they mark an intermediate compartment of PSII assembly. These results reveal a novel chloroplast compartment that houses de novo PSII biogenesis and the regulated transport of newly assembled PSII complexes to thylakoid membranes throughout the chloroplast.

Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii

“…1C, compare fractions 7-9 and fractions 10-12) (22). Finally, the mRNA encoding PsaAp was not recruited to the Tzone under the same conditions that recruited two PSII subunit mRNAs (6). Taken together, these results suggest that PSI subunit synthesis and assembly occur at thylakoids, and not CTM.…”

“…First, it is specific to import machinery because it was not observed for many other chloroplast proteins, the localization of which we have examined with this method (6,7,23). Second, this localization pattern may be physiologically relevant because the percentage of cells showing it dropped during incubation in the dark for 2 h, a condition associated with reduced rates of PSII biogenesis and chloroplast protein import in C. reinhardtii (6,24). When ML cells were dark-adapted immediately before fixation, the percentages showing localization around lobe junctions dropped from 48 to 11% for Toc75 (n = 74) and from 47 to 15% for Tic110 (n = 52).…”

“…An alternative model proposes that a specific "translation zone" (T-zone) in the chloroplast of the green alga Chlamydomonas reinhardtii is a privileged site of protein synthesis for the de novo biogenesis of PSII and possibly other complexes (6,7). This T-zone was defined by the colocalization of markers of the chloroplast ribosome, chloroplast mRNAs encoding PSII subunits, and the PSII translation factor RBP40 [RBP40 is also called RB38 (8,9)], as seen by confocal microscopy (6).…”

“…We postulated that the T-zone contains a chloroplast translation membrane (CTM) as a privileged site for the synthesis of PSII subunits encoded by the chloroplast genome because chloroplasts ribosomes synthesizing PSII subunits are bound to membrane, but the chloroplast envelope and most thylakoid membranes are outside the T-zone (6,10). This prediction provided an avenue to test the T-zone model at the biochemical level.…”

“…This scheme resolves chloroplast membranes with the high density of rough ER membrane (11) because we predicted that a CTM would also have a high-density membrane as a result of its having bound ribosomes. We focused on the location of PSII subunit synthesis because the evidence for the T-zone was established for chloroplast mRNAs encoding PSII subunits (6). We also identified unique domains of chloroplast envelope that are enriched in the translocons of the outer and inner chloroplast envelope membranes (TOC-TIC) and located adjacent to cytosolic regions where previous in situ evidence supports the localized translation of the mRNA encoding a subunit of the light harvesting complex II (LHCII), which is peripherally associated with PSII (7).…”