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

Abstract

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.

Stepwise evolution of supercomplex formation with photosystem I is required for stabilization of chloroplast NADH dehydrogenase‐like complex: Lhca5‐dependent supercomplex formation in Physcomitrella patens

“…In contrast, the stromal loop is divergent among LHCI family members, even in the same species. In Arabidopsis, the stromal loop of Lhca6 is necessary for linker function and is sufficient for conferring linker function on Lhca2, from which Lhca6 originates (Otani et al, 2017). Because of the constraints on interaction with the PSI core or neighbouring LHCIs, other domains of LHCI must be highly conserved.…”

“…In contrast, the stromal loop is highly conserved in Lhca5 between Physcomitrella and Arabidopsis, implying that this region contributes to the function of Lhca5. Notably, the stromal loop of Lhca6 is required, and is sufficient, for the linker function for NDH-PSI supercomplex formation in Arabidopsis (Otani et al, 2017).…”

“…LHCI proteins have three transmembrane helices (helices B, C, and A); the loops between helices B and C are exposed to the lumen, and those between C and A (stromal loop) are exposed to the stroma (Qin et al, 2015). The stromal loop in Lhca6 is required, and is sufficient, for the linker function in Arabidopsis (Otani et al, 2017).…”

Specific substitutions of light‐harvesting complex I proteins associated with photosystem I are required for supercomplex formation with chloroplast NADH dehydrogenase‐like complex

“…Lhca2 is absent in the partial NDH-PSI 1L supercomplex in the lhca5 mutant Because of the sequence similarity between Lhca2 and Lhca6 (Otani et al, 2017), Lhca6 may substitute for Lhca2 in the NDH-PSI supercomplex. Alternatively, Lhca6 may be localized to the outside of the intact LHCI arc consisting of Lhca1 to Lhca4 to intermediate supercomplex formation.…”

“…In our working model, the linker proteins may substitute for the major LHCI proteins in the NDH-PSI supercomplex. The model is especially probable between Lhca2 and Lhca6 because of their high sequence and functional similarity (Otani et al, 2017). It is still possible that each copy of PSI-LHCI contains a full set of major LHCI tetramers and that the linker proteins are outside the LHCI arc, although it is unlikely that the single-particle images support this idea.…”