In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System

Abstract

Using a homologous chloroplast translation system, we have reconstituted insertion and processing of the chloroplast-encoded thylakoid protein cytochrome f (pCytf). Cross-linking demonstrated that pCytf nascent chains when attached to the 70 S ribosome tightly interact with cpSecA, but this is strictly dependent on thylakoid membranes and a functional signal peptide. This indicates that cpSecA is only operative in pCytf biogenesis when it is bound to the membrane, most likely as part of the Sec translocon. No evidence for interaction between the 54-kDa subunit of the chloroplast signal recognition particle (cpSRP) and the pCytf nascent chain could be detected, suggesting that pCytf, in contrast to the polytopic D1 protein, does not require cpSRP for targeting. Insertion of pCytf occurred only co-translationally, resulting in processing and accumulation of both the processed signal peptide and the mature protein in the thylakoid. This co-translational membrane insertion and processing required a functional signal peptide and was inhibited by azide, demonstrating that cpSecA is essential for translocation of the soluble luminal domain. pCytf also associated posttranslationally with thylakoids, but the soluble N-terminal domain could not be translocated into the lumen. This is the first study in which synthesis, targeting, and insertion of a chloroplast-encoded thylakoid membrane protein is reconstituted from exogenous transcripts and using the chloroplast translational machinery.

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

“…2B) either as a ratio (upper plot) or separately (lower plot). All ORFs encoding proteins that were shown previously to integrate cotranslationally into the thylakoid membrane (PsbA, PsbB, PsbC, PsbD, PsaA PsaB, and PetA) (10)(11)(12)(13)22) are represented by prominent membrane-associated peaks, validating the method. Additional peaks revealed the thylakoid-associated translation of 12 proteins that had not been assayed in prior studies: AtpF, AtpI, PetB, NdhA, NdhB, NdhC, NdhD, NdhE, NdhF, NdhG, Ycf4, and CcsA (Fig.…”

“…CemA is the sole plastidencoded protein in maize that localizes to the inner envelope membrane and is discussed below. PetA is the sole plastidencoded protein harboring a cleavable N-terminal cpSecAdependent signal sequence, which mediates its targeting to the thylakoid membrane (12,15,16). The petA ORF engages the membrane after synthesis of ∼100 amino acids (Fig.…”

“…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).…”

“…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). In vitro cross-linking experiments showed further that nascent PsbA is in proximity to both cpSRP54 (18) and cpSecY (19).…”

Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast‐encoded D1 protein; ‘cpSRP54 caught in the act’

“…D1 polypeptide Full-length D1 protein synthesized from exogenous psbA transcripts in the homologous pea chloroplast translation system (with the complete 5P untranslated region) accumulated stable to relatively high levels, as compared to other plastidencoded gene products such as cytochrome f [19] and CFo-III (Rohl and van Wijk, unpublished) ( Fig. 1).…”