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 b6 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 b6 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.