HCF164 Receives Reducing Equivalents from Stromal Thioredoxin across the Thylakoid Membrane and Mediates Reduction of Target Proteins in the Thylakoid Lumen

Abstract

HCF164 is a membrane-anchored thioredoxin-like protein known to be indispensable for assembly of cytochrome b 6 f in the thylakoid membranes. In this study, we report the finding that chloroplast stroma m-type thioredoxin is the source of reducing equivalents for reduction of HCF164 in the thylakoid lumen, providing strong evidence that higher plant chloroplasts possess a trans-membrane reducing equivalent transfer system similar to that found in bacteria. To probe the function of HCF164 in the lumen, a screen to identify the reducing equivalent acceptor proteins of HCF164 was carried out by using a resin-immobilized HCF164 single cysteine mutant, leading to the isolation of putative target thylakoid proteins. Among the newly identified target proteins, the reduction of the PSI-N subunit of photosystem I by HCF164 was confirmed both in vitro and in isolated thylakoids. Two components of the cytochrome b 6 f complex, the cytochrome f and Rieske FeS proteins, were also identified as novel potential target proteins. The data presented here suggest that HCF164 serves as an important transducer of reducing equivalents to proteins in the thylakoid lumen.The redox state of higher plant chloroplasts undergoes significant fluctuations in both light and dark conditions. In the light, photosynthetic electron transport via ferredoxin and ferredoxin-NADP ϩ reductase (FNR) 2 results in the synthesis of NADPH, which is used as a source of reducing equivalents for carbon fixation. However, a portion of the electrons produced by the photosynthetic electron transport is transferred to thioredoxin (Trx) through ferredoxin and ferredoxin-thioredoxin reductase (1-3). The two stromal thioredoxin isoforms Trx-f and Trx-m were originally identified for this pathway (4, 5); they have been extensively characterized and are known to regulate the activity of a number of thiol enzymes by alteration of their redox state. Completion of the Arabidopsis genomic data base showed that higher plants possess a number of Trx isoforms in chloroplasts, mitochondria, and in the cytosol (6 -8), although their specific localization and function within these cellular compartments have yet to be determined (9, 10).In recent years, highly effective screening methods have been developed that have lead to the isolation of a large number and variety of higher plant Trx-target protein candidates (11)(12)(13)(14)(15)(16). This has allowed the characterization of a wide variety of previously unknown Trx-regulated enzymes in the chloroplast stroma, for example (6 -8, 12, 17, 18). In contrast, our knowledge of the thiol-disulfide redox control system in the thylakoid lumen has been limited so far (19). Meurer et al. identified mutants of Arabidopsis that displayed a high chlorophyll fluorescence phenotype (hcf mutants) under standard photosynthetic conditions (20, 21), suggesting a defect in photosynthetic electron transport. Among these, the hcf164 mutant was found to be impaired in the stable assembly of the cytochrome b 6 f complex within thylakoid membranes (22...

Arabidopsis Cotyledon-Specific Chloroplast Biogenesis Factor CYO1 Is a Protein Disulfide Isomerase

“…Recently, Motohashi and Hisabori (2006) reported that several thylakoid membrane proteins interact with HCF167, a thioredoxinlike protein in thylakoids. All of the proteins they identified, including cytochrome f, Rieske FeS protein, PSI-N, LHCB5, FTSH2, FTSH8, and three CF 1 subunits of ATPase, have multiple Cys residues.…”

“…To analyze the topology of CYO1 in the thylakoid membrane, we performed a protease protection assay (Motohashi and Hisabori, 2006). Incubation of sonicated thylakoid membrane samples with the protease thermolysin resulted in the degradation of FNR, which is located on the stroma-exposed surface of thylakoid membranes, and PSI-N (for the N subunit of photosystem I), which is located on the lumen-exposed surface of the thylakoid membrane.…”

“…After incubation for 1 h on ice, samples were centrifuged at 10,000g for 10 min at 08C to separate soluble and insoluble fractions. A protease protection assay was performed as described (Motohashi and Hisabori, 2006). Blue native gel electrophoresis was performed as described (Asakura et al, 2004) (see also http://www.hos.ufl.edu/clineweb/BNgel.htm) with the following modifications.…”

Redox Regulation of Rotation of the Cyanobacterial F1-ATPase Containing Thiol Regulation Switch

“…Other Proteins-Recombinant thioredoxin-f of S. oleracea was expressed in E. coli and purified as described (23). Chloroplast F 1 -ATPase (␣ 3 ␤ 3 ␥␦⑀) was purified from fresh market spinach leaves (24) and the ␦ and ⑀ subunits were removed by the following procedures.…”

Thioredoxin-mediated reduction of the photosystem I subunit PsaF and activation through oxidation by the interaction partner plastocyanin

“…However, little is known about Trx-regulation in the lumen. It is only recently that a Trx-like protein was found within the lumen, leading to a new field of exploration [7]. In that study, it was shown that the protein HCF164 can reduce Cys residues of the PSI subunit PsaN.…”