Abstract
Chloroplast genomes encode ∼ 37 proteins that integrate into the thylakoid membrane. The mechanisms that target these proteins to the membrane are largely unexplored. We used ribosome profiling to provide a comprehensive, high-resolution map of ribosome positions on chloroplast mRNAs in separated membrane and soluble fractions in maize seedlings. The results show that translation invariably initiates off the thylakoid membrane and that ribosomes synthesizing a subset of membrane proteins subsequently become attached to the membrane in a nuclease-resistant fashion. The transition from soluble to membrane-attached ribosomes occurs shortly after the first transmembrane segment in the nascent peptide has emerged from the ribosome. Membrane proteins whose translation terminates before emergence of a transmembrane segment are translated in the stroma and targeted to the membrane posttranslationally. These results indicate that the first transmembrane segment generally comprises the signal that links ribosomes to thylakoid membranes for cotranslational integration. The sole exception is cytochrome f, whose cleavable N-terminal cpSecA-dependent signal sequence engages the thylakoid membrane cotranslationally. The distinct behavior of ribosomes synthesizing the inner envelope protein CemA indicates that sorting signals for the thylakoid and envelope membranes are distinguished cotranslationally. In addition, the fractionation behavior of ribosomes in polycistronic transcription units encoding both membrane and soluble proteins adds to the evidence that the removal of upstream ORFs by RNA processing is not typically required for the translation of internal genes in polycistronic chloroplast mRNAs.
Highlights
Chloroplast genomes encode ∼37 proteins that integrate into the thylakoid membrane
Leaf homogenates were initially treated with micrococcal nuclease to release ribosomes from membranes that were tethered only by mRNA; this treatment will release ribosomes that are bound to membranes due to their presence on an mRNA that is membranetethered via a different ribosome or via an RNA binding protein
Those membrane proteins whose translation terminates before exposure of one of these signals are translated off the membrane and must be posttranslationally targeted
Summary
Chloroplast genomes encode ∼37 proteins that integrate into the thylakoid membrane. The mechanisms that target these proteins to the membrane are largely unexplored. Membrane proteins whose translation terminates before emergence of a transmembrane segment are translated in the stroma and targeted to the membrane posttranslationally These results indicate that the first transmembrane segment generally comprises the signal that links ribosomes to thylakoid membranes for cotranslational integration. Intensive study of the mechanisms underlying the thylakoid localization of nucleus-encoded proteins revealed the participation of four machineries of cyanobacterial ancestry: the cpSec, cpTAT, cpSRP, and ALB3 systems In vitro cross-linking experiments showed further that nascent PsbA is in proximity to both cpSRP54 [18] and cpSecY [19] It is not known whether the majority of chloroplast-encoded thylakoid proteins are co- or posttranslationally integrated, nor is it known which, if any, of the known thylakoid targeting machineries are involved in their targeting and integration. We adapted this method for the rapid analysis of chloroplast translation by substituting highresolution tiling microarrays for the deep-sequencing step [21]
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