Translation and Co-translational Membrane Engagement of Plastid-encoded Chlorophyll-binding Proteins Are Not Influenced by Chlorophyll Availability in Maize

Abstract

Chlorophyll is an indispensable constituent of the photosynthetic machinery in green organisms. Bound by apoproteins of photosystems I and II, chlorophyll performs light-harvesting and charge separation. Due to the phototoxic nature of free chlorophyll and its precursors, chlorophyll synthesis is regulated to comply with the availability of nascent chlorophyll-binding apoproteins. Conversely, the synthesis and co-translational insertion of such proteins into the thylakoid membrane have been suggested to be influenced by chlorophyll availability. In this study, we addressed these hypotheses by using ribosome profiling to examine the synthesis and membrane targeting of chlorophyll-binding apoproteins in chlorophyll-deficient chlH maize mutants (Zm-chlH). ChlH encodes the H subunit of the magnesium chelatase (also known as GUN5), which catalyzes the first committed step in chlorophyll synthesis. Our results show that the number and distribution of ribosomes on plastid mRNAs encoding chlorophyll-binding apoproteins are not substantially altered in Zm-chlH mutants, suggesting that chlorophyll has no impact on ribosome dynamics. Additionally, a Zm-chlH mutation does not change the amino acid position at which nascent chlorophyll-binding apoproteins engage the thylakoid membrane, nor the efficiency with which membrane-engagement occurs. Together, these results provide evidence that chlorophyll availability does not selectively activate the translation of plastid mRNAs encoding chlorophyll apoproteins. Our results imply that co- or post-translational proteolysis of apoproteins is the primary mechanism that adjusts apoprotein abundance to chlorophyll availability in plants.

Chloroplast Translation: Structural and Functional Organization, Operational Control, and Regulation

“…Although pulse labeling can reveal protein synthesis rates, it cannot unambiguously distinguish between the absence of synthesis and rapid degradation of newly synthesized proteins (especially not for proteins with high turnover rates, such as PsbA). A recent ribosome profiling analysis of plastid translation in a maize mutant with knocked-out chlorophyll synthesis showed that the synthesis of plastid-encoded chlorophyll binding apoproteins is virtually unaltered in the absence of chlorophyll (Zoschke et al, 2017). Even the cotranslational thylakoid membrane engagement of nascent apoproteins was shown to be independent of chlorophyll synthesis (Zoschke et al, 2017).…”

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

“…To elucidate the role of ribosome-bound cpSRP54 in translation or cotranslational targeting of plastid-encoded thylakoid membrane proteins, a previously described ribosomal profiling approach was used that distinguishes between membrane-associated and soluble footprints and identifies the regions of cotranslational membrane engagement in nascent peptides (Zoschke and Barkan, 2015;Zoschke et al, 2017). To this end, ribosome footprints from separated (thylakoid) membrane and soluble (stroma) fractions of the wild-type Arabidopsis plants and a mutant lacking cpSRP54 (ffc; Amin et al, 1999) were isolated and analyzed by hybridization to microarrays with highly tiled probes covering all chloroplast reading frames.…”

Pausing of Chloroplast Ribosomes Is Induced by Multiple Features and Is Linked to the Assembly of Photosynthetic Complexes

“…We cannot distinguish whether this is caused by features of the mRNA and/or nascent chain 322 or is itself dependent on the incorporation of cofactors. A recent analysis found no changes in translation 323 elongation in mutants of chlorophyll synthesis, indicating that at least chlorophyll supply does not influence 324 ribosome pausing (Zoschke et al, 2017). It is known that Mn 4 CaO 5 cluster binds to D1 and CP43.…”