A novel serine/proline‐rich domain in combination with a transmembrane domain is required for the insertion of AtTic40 into the inner envelope membrane of chloroplasts

Abstract

SummaryAtTic40 is part of the chloroplastic protein import apparatus that is anchored in the inner envelope membrane by a single N-terminal transmembrane domain, and has a topology in which the bulk of the C-terminal domain is oriented toward the stroma. The targeting of AtTic40 to the inner envelope membrane involves two steps. Using an in vitro import assay, we showed that the sorting of AtTic40 requires a bipartite transit peptide, which was first cleaved by the stromal processing peptidase (SPP), thus generating a soluble AtTic40 stromal intermediate (iAtTic40). iAtTic40 was further processed by a second unknown peptidase, which generates its mature form (mAtTic40). Using deletion mutants, we identified a sequence motif N-terminal of the transmembrane domain that was essential for reinsertion of iAtTic40 into the inner envelope membrane. We have designated this region a serine/proline-rich (S/P-rich) domain and present a model describing its role in the targeting of AtTic40 to the inner envelope membrane.

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

“…Some nucleus-encoded envelope proteins are imported into the stroma and subsequently "exported" to the inner envelope (46). TIC40, a well-studied example, has a serine/ proline-rich domain that is crucial for inner envelope targeting (47). When TIC40 was expressed from a chloroplast transgene, it was targeted to the inner envelope (48), suggesting that similar mechanisms can target nucleus-and plastid-encoded proteins to the inner envelope.…”

Plastids Contain a Second Sec Translocase System with Essential Functions

“…Several nucleus-encoded envelope proteins are candidate SCY2 substrates. TIC40 and TIC110 are transiently present in the stroma as soluble intermediates and integrated into the inner envelope membrane, possibly by a conservative sorting pathway of unknown identity (Li and Schnell, 2006;Tripp et al, 2007). Although soluble intermediates of TIC21 and TIC110 accumulate in null Tic40 mutants, TIC40 is thought to play an accessory, rather than a central, role, because translocation is slowed but not blocked (Chiu and Li, 2008).…”

“…The signals and systems involved in targeting to the thylakoid membranes and lumen are relatively well studied and show clear homologies with bacterial transport systems (for review, see Schü nemann, 2007;Cline and DabneySmith, 2008). Although it has been clearly established that certain inner membrane proteins, most notably TIC21, TIC40, and TIC110, also have soluble stromal intermediates (Li and Schnell, 2006;Tripp et al, 2007;Vojta et al, 2007;Chiu and Li, 2008) and therefore require a postimport pathway for integration, a translocase that mediates insertion into the inner membrane or translocation to the intramembrane space has not been identified (Tripp et al, 2007).…”

Chaperone-assisted Post-translational Transport of Plastidic Type I Signal Peptidase 1

“…pIVTGW-SP6 was generated by ligating a 4-kb fragment of pCRII-TOPO (Life Technologies, Inc.) and a 1.8-kb fragment of pMDC32 (25) after digesting each plasmid with KpnI and SacI, followed by insertion of an oligonucleotide linker into the AscI-KpnI site to remove an in-frame start codon upstream of the cloning site. Plasmids encoding Pisum sativum (pea) Tic22 (26), Arabidopsis Tic40 (27), and pea LHCP (28) were as described previously. Arabidopsis OE23 (U19899), OE33 (U13665), and PsbW (U12467) in pUNI51 were obtained from the Arabidopsis Biological Resource Center (Columbus, OH).…”