Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex1

Abstract

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O 2 -evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two proteintranslocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.Although the chloroplast possesses its own genome, the majority of proteins found in this organelle are nuclear encoded, synthesized on cytoplasmic ribosomes, and posttranslationally imported. The process by which these proteins are directed to their final destination is complicated by the fact that chloroplasts possess three membranes that define six distinct destinations for the newly imported protein.Nuclear-encoded chloroplast proteins are generally synthesized as higher-molecular-mass precursors that possess a cleavable, amino-terminal extension called a transit peptide. This topogenic sequence acts to direct the polypeptide from the cytoplasm to its final location within the chloroplast. Proteins residing in the thylakoid lumen are required to cross the envelope and thylakoid membranes, and generally possess a bipartite transit peptide. The first region of the targeting sequence directs the protein across the envelope membranes into the stroma, where it is cleaved by the stromal-processing protease, resulting in an intermediatesized protein species. The remaining region of the transit peptide then targets this stromal intermediate to the thylakoid lumen, where it is removed by the membranebound lumenal processing protease, generating the mature-sized protein (for review, see Theg and Scott, 1993;Cline and Henry, 1996;Haucke and Schatz, 1997).There have been extensive efforts to determine the mechanism by which proteins are transported into or across the thylakoid membrane. Currently, four pathways have been defined by their energy requirements, and some of their components have been identified. The first pathway appears to be spontaneous; no energy sources or proteasesensitive membrane factors seem to be required (Michl et al....

Role of Vesicle‐Inducing Protein in Plastids 1 in cpTat transport at the thylakoid

“…Thus, the reactions were performed in long microfuge tubes with three layers: the transport reaction at the top, a layer of insulating silicone oil in the middle (Hurwitz et al. , 1965; Leheny et al. , 1998), and a bottom layer containing 20% TCA (transport and binding assays) or 2 × IB (chase assays).…”

An Essential Role for the DnaK Molecular Chaperone in Stabilizing Over-expressed Substrate Proteins of the Bacterial Twin-arginine Translocation Pathway

“…DnaK, GroEL) and proteases (e.g. ClpXP, FtsH) play an important role in the early stages of Tat targeting and translocation in bacteria 25,36,38,39,45 and plants [55][56][57][58] (for a review see Fisher and DeLisa 34 ). Accordingly, we tested a panel of E. coli mutant strains lacking well-characterized chaperones or proteases and found that the molecular chaperone DnaK exerted the most significant effect on the expression and transport of synthetic Tat substrates such as ssTorA-GFP-SsrA, ssTorA-GFP and ssTorA-MBP.…”

Polyphenol oxidase can cross thylakoids by both the Tat and the Sec‐dependent pathways: a putative role for two stromal processing sites

“…Furthermore, the fact that import of Tat proteins takes place even when the trans ‐thylakoid ΔpH is depleted (Di Cola et al 2005a, Finazzi et al 2003), limits the use of nigericin as a pathway‐specific inhibitor. Azide was shown to inhibit the import of OE17, a Tat pathway protein, but unlike pPPO the effect was seen only after pOE17 import was inhibited by nigericin (Leheny et al 1998).…”