Evaluating the Functional Pore Size of Chloroplast TOC and TIC Protein Translocons

Abstract

The degree of residual structure retained by proteins while passing through biological membranes is a fundamental mechanistic question of protein translocation. Proteins are generally thought to be unfolded while transported through canonical proteinaceous translocons, which has historically been the thought for the translocons of the outer and inner chloroplast envelope membranes (TOC and TIC). Here, we readdressed the issue and found that medium-sized tightly folded proteins such as the 22 kDa dihydrofolate reductase (DHFR) can be tolerated by TOC and TIC. Chimeric DHFR fused with RuBisCO small subunit transit peptide (tp22DHFR) was found to be imported into chloroplasts in complex with its stabilizing ligand, methotrexate (MTX), in a folded conformation. Following import, both mature tp22DHFR and MTX were found in the chloroplast stroma. A subsaturating concentration of MTX was used to exclude the possibility that MTX was stripped off tp22DHFR, independently imported into the chloroplasts, and reassociated with imported tp22DHFR. Independent MTX import was further excluded by use of fluorescein conjugated MTX (FMTX), which has very slow membrane transport rates relative to unconjugated MTX. The TOC/TIC pore size was determined by probing the translocons with particles of fixed diameter and found to be greater than 25.6 [A], large enough to support folded DHFR import. The pore size is also larger than those of the mitochondrial protein translocons that have a requirement for protein unfolding.\n\nSIGNIFICANCEThe chloroplast TOC and TIC translocons are responsible for the import of up to 95% of all chloroplast proteins and are therefore essential for plastid biogenesis and photosynthesis. However, the mechanisms of protein import into chloroplasts are not well understood. The TOC/TIC translocons have long been suggested to have a strong unfoldase activity relative to other comparable protein translocons. Here, we present data suggesting that this is not true, and that instead, they possess a relatively large pore size. This identifies TOC and TIC as rather unique protein translocons capable of transporting folded proteins across a double membrane barrier, which has important implications in the mechanisms of TOC/TIC function and biogenesis of photosynthetic proteins.\n\nClassification - Biochemistry