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 mechanismsposttranslational, 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.chloroplast ͉ FISH ͉ organelle ͉ protein targeting ͉ translation

Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii

“…The effect of light on the relocalization of the TIC and TOC import machinery to lobe junctions might be relevant to the light stimulation of chloroplast protein import which has been observed in C. reinhardtii and vascular plants (24,30). Moreover, the Rubisco holoenzyme might be assembled in the pathways described here because its small subunit is imported via the TOC and TIC pathway and the chloroplast mRNA encoding the large subunit localizes in situ in the T-zone and is translated in association with membrane (7,31,32). Our findings build upon growing evidence of complex cytological organizations of biogenic processes in organelles and bacteria.…”

“…In the Tzone, the CTM is a platform for the synthesis of the plastid genomeencoded subunits. Chloroplast ribosome subunits and PSII subunit mRNAs are recruited to membranes in the T-zone by translation independent mechanisms (e.g., tethering by membrane bound RNA-binding proteins of LDM) (7). The particularly high affinity with which the 30S subunit is bound to the CTM (Fig.…”

“…This pattern is interesting for three reasons. 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).…”

“…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).…”

Genome-wide analysis of thylakoid-bound ribosomes in maize reveals principles of cotranslational targeting to the thylakoid membrane

“…Pioneering studies demonstrated that some chloroplast ribosomes are attached to the thylakoid membrane by the nascent peptide, implying a cotranslational integration mechanism (8,9). Several specific plastid-encoded proteins have been shown to integrate cotranslationally: the PSII subunits PsbA (also known as D1), PsbB, PsbC, and PsbD; the PSI subunits PsaA and PsaB; and the cytochrome b 6 f subunit PetA (also known as cytochrome f) (10)(11)(12)(13)(14). The insertion of PetA into the membrane requires cpSecA (12,15,16), whereas PsbA integrates independent of both the cpSecA and cpTAT systems (17).…”