Ribosome-associated chloroplast SRP54 enables efficient co-translational membrane insertion of key photosynthetic proteins

Abstract

Key proteins of the photosynthetic complexes are encoded in the chloroplast genome and cotranslationally inserted into the thylakoid membrane. However, the molecular details of this process are largely unknown. Here, we demonstrate by ribosome profiling that the conserved chloroplast signal recognition particle subunit (cpSRP54) is required for efficient cotranslational targeting of several central photosynthetic proteins, such as the PSII PsbA (D1) subunit, in Arabidopsis (Arabidopsis thaliana). High-resolution analysis of membrane-associated and soluble ribosome footprints revealed that the SRP-dependent membrane targeting of PsbA is already initiated at an early translation step before exposure of the nascent chain from the ribosome. In contrast to cytosolic SRP, which contacts the ribosome close to the peptide tunnel exit site, analysis of the cpSRP54/ribosome binding interface revealed a direct interaction of cpSRP54 and the ribosomal subunit uL4, which is not located at the tunnel exit site but forms a part of the internal peptide tunnel wall by a loop domain. The plastid-specific C-terminal tail region of cpSRP54 plays a crucial role in uL4 binding. Our data indicate a novel mechanism of SRP-dependent membrane protein transport with the cpSRP54/uL4 interaction as a central element in early initiation of cotranslational membrane targeting.

Co-Translational Protein Folding and Sorting in Chloroplasts

“…While the post-translational targeting pathway of cpSRP54 and cpSRP43 is relatively well understood, reviewed in [106], rather little is known about the co-translational function and the mechanism of target protein integration into thylakoid membranes. At least in Pisum sativum, binding to the ribosome is proposed to occur via the M domain interacting with the surface-exposed part of uL4c, a ribosomal protein of the large subunit ( Figure 2) [107]. For many years cpSRP54 was only known to co-translationally associate with the photosystem II (PS II) core subunit PsbA and the cytochrome b 6 f complex subunit PetB [108][109][110].…”

“…For many years cpSRP54 was only known to co-translationally associate with the photosystem II (PS II) core subunit PsbA and the cytochrome b 6 f complex subunit PetB [108][109][110]. An elegant recent ribosome profiling study comparing thylakoid association of translating ribosomes in A. thaliana wild-type and srp54 mutant lines greatly extending the pool of putative cpSRP54 substrates [107]. This ribosome profiling approach showed that the loss of cpSRP54 does not generally result in different footprint yields or altered translational output of all plastid open reading frames (ORFs), but there was a clear decrease in membrane protein footprint yield of putative substrate proteins, showing the Plants 2020, 9, 214 7 of 15 importance of cpSRP54 for protein integration into thylakoid membranes.…”

“…This ribosome profiling approach showed that the loss of cpSRP54 does not generally result in different footprint yields or altered translational output of all plastid open reading frames (ORFs), but there was a clear decrease in membrane protein footprint yield of putative substrate proteins, showing the Plants 2020, 9, 214 7 of 15 importance of cpSRP54 for protein integration into thylakoid membranes. Among the putative nascent polypeptides that are co-translationally targeted to thylakoid membranes are the PS I core subunits PsaA and PsaB, the PS II subunits PsbA, PsbB and PsbD, the above mentioned cytochrome b 6 f complex subunit PetB and the NADH dehydrogenase-like complex subunit NdhD [107]. This goes in hand with the previous observation that chloroplast protein synthesis of nascent integral thylakoid proteins seems to start within the soluble stroma and ribosomes are translocated to thylakoid membranes once the first transmembrane segment emerges from the ribosomal exit tunnel [111].…”

“…Plants 2020, 9, sativum, binding to the ribosome is proposed to occur via the M domain interacting with the surfaceexposed part of uL4c, a ribosomal protein of the large subunit ( Figure 2) [107]. For many years cpSRP54 was only known to co-translationally associate with the photosystem II (PS II) core subunit PsbA and the cytochrome b6f complex subunit PetB [108][109][110].…”

“…Most importantly, a structural hairpin is absent, which would circumvent the binding of cpSRP54 if the interaction was of similar nature as observed in bacteria [52,127,128]. Interestingly, it was recently shown that the ribosomal interaction of cpSRP54 rather involves uL4c instead of uL23c [107]. This interaction utilizes the hydrophobic groove and the C-terminal tail region of the M domain of cpSRP54 to bind to the globular part of uL4c, which is exposed to the surface.…”