Stromal Loop of Lhca6 is Responsible for the Linker Function Required for the NDH-PSI Supercomplex Formation.

The light-harvesting complex I (LHCI) proteins in Arabidopsis thaliana are encoded by six genes. Major LHCI proteins (Lhca1-Lhca4) harvest light energy and transfer the resulting excitation energy to the PSI core by forming a PSI supercomplex. In contrast, the minor LHCI proteins Lhca5 and Lhca6 contribute to supercomplex formation between the PSI supercomplex and the chloroplast NADH dehydrogenase-like (NDH) complex, although Lhca5 is also solely associated with the PSI supercomplex. Lhca6 was branched from Lhca2 during the evolution of land plants. In this study, we focused on the molecular evolution involved in the transition from a major LHCI, Lhca2, to the linker protein Lhca6. To elucidate the domains of Lhca6 responsible for linker function, we systematically swapped domains between the two LHCI proteins. To overcome problems due to the low stability of chimeric proteins, we employed sensitive methods to evaluate supercomplex formation: we monitored NDH activity by using Chl fluorescence analysis and detected NDH-PSI supercomplex formation by using protein blot analysis in the form of two-dimensional blue-native (BN)/SDS-PAGE. The stromal loop of Lhca6 was shown to be necessary and sufficient for linker function. Chimeric Lhca6, in which the stromal loop was substituted by that of Lhca2, was not functional as a linker and was detected at the position of the PSI supercomplex in the BN-polyacrylamide gel. The stromal loop of Lhca6 is likely to be necessary for the interaction with chloroplast NDH, rather than for the association with the PSI supercomplex.