Multiple fates of non‐mature lumenal proteins in thylakoids

Abstract

SUMMARYMost proteins found in the thylakoid lumen are synthesized in the cytosol with an N-terminal extension consisting of transient signals for chloroplast import and thylakoid transfer in tandem. The thylakoid-transfer signal is required for protein sorting from the stroma to thylakoids, mainly via the cpSEC or cpTAT pathway, and is removed by the thylakoidal processing peptidase in the lumen. An Arabidopsis mutant lacking one of the thylakoidal processing peptidase homologs, Plsp1, contains plastids with anomalous thylakoids and is seedling-lethal. Furthermore, the mutant plastids accumulate two cpSEC substrates (PsbO and PetE) and one cpTAT substrate (PsbP) as intermediate forms. These properties of plsp1-null plastids suggest that complete maturation of lumenal proteins is a critical step for proper thylakoid assembly. Here we tested the effects of inhibition of thylakoid-transfer signal removal on protein targeting and accumulation by examining the localization of non-mature lumenal proteins in the Arabidopsis plsp1-null mutant and performing a protein import assay using pea chloroplasts. In plsp1-null plastids, the two cpSEC substrates were shown to be tightly associated with the membrane, while non-mature PsbP was found in the stroma. The import assay revealed that inhibition of thylakoid-transfer signal removal did not disrupt cpSEC-and cpTAT-dependent translocation, but prevented release of proteins from the membrane. Interestingly, non-mature PetE2 was quickly degraded under light, and unprocessed PsbO1 and PsbP1 were found in a 440-kDa complex and as a monomer, respectively. These results indicate that the cpTAT pathway may be disrupted in the plsp1-null mutant, and that there are multiple mechanisms to control unprocessed lumenal proteins in thylakoids.

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments

“…The PC degradation activity must be metal independent but is dependent on stromal proteins, the proton motive force across the thylakoid, and ATP hydrolysis. Thylakoid-retained PC cleavage intermediates are exposed in part to the stroma (Midorikawa and Inoue, 2013), potentially allowing the access of stromal proteases, as in the case of PAA2/HMA8 for Clp (Tapken et al, 2015). The PsbP intermediate is detected as a monomer in the stroma when Plsp1 is missing (Shipman-Roston et al, 2010;Midorikawa and Inoue, 2013) but exists within the membrane, presumably through an association with the TAT machinery, when its TPP processing site is abolished (Frielingsdorf and Klösgen, 2007;Midorikawa and Inoue, 2013).…”

“…Thylakoid-retained PC cleavage intermediates are exposed in part to the stroma (Midorikawa and Inoue, 2013), potentially allowing the access of stromal proteases, as in the case of PAA2/HMA8 for Clp (Tapken et al, 2015). The PsbP intermediate is detected as a monomer in the stroma when Plsp1 is missing (Shipman-Roston et al, 2010;Midorikawa and Inoue, 2013) but exists within the membrane, presumably through an association with the TAT machinery, when its TPP processing site is abolished (Frielingsdorf and Klösgen, 2007;Midorikawa and Inoue, 2013). An earlier report has shown that TAT-dependent substrates can be returned to the stroma and that disruption of the TPP sites of the TAT substrates causes their unprocessed intermediates to accumulate in the thylakoids, where they are likely to be trapped in the sorting machinery (Di Cola and Robinson, 2005).…”

“…An earlier report has shown that TAT-dependent substrates can be returned to the stroma and that disruption of the TPP sites of the TAT substrates causes their unprocessed intermediates to accumulate in the thylakoids, where they are likely to be trapped in the sorting machinery (Di Cola and Robinson, 2005). A possible function of thylakoid Plsp1 in preventing reverse translocation has been proposed (Midorikawa and Inoue, 2013). Interestingly, a lumenal protein lacking the lumenal target peptide is prone to mislocalization in stroma, where it is readily degraded (Halperin and Adam, 1996).…”

“…Knockout of Plsp1 causes the accumulation of processing intermediates of the SEC substrates, PC and OE33/PsbO, and the TAT substrate, OE23/PsbP (Shipman-Roston et al, 2010;Albiniak et al, 2012). The absence of Plsp1 and the elimination of TPP cleavage sites seem not to affect the SEC pathway itself but impede substrate release from the membrane; indeed, PC and PsbO intermediates are stuck in the thylakoid membrane despite their proper membrane targeting (Shackleton and Robinson, 1991;Frielingsdorf and Klösgen, 2007;Midorikawa and Inoue, 2013). It is noteworthy that precursor PsbO is stably present in a 440-kD complex that is not fully characterized but is redox sensitive, protease tolerant, and distinct from the PSII or SEC machinery, whereas unprocessed PC is prone to light-driven proteolysis by unknown protease(s).…”

“…It is noteworthy that some of the ClpS1 targets isolated through affinity purification are metal-binding or metal-related proteins (Nishimura et al, 2013). sorting requires TTS, which must be removed by TPP for protein assembly, posttranslocational release from the membrane, and, therefore, proper thylakoid formation (Inoue et al, 2005;Shipman-Roston et al, 2010;Midorikawa and Inoue, 2013).…”