Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley

Abstract

The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).

LIL3, a Light-Harvesting Complex Protein, Links Terpenoid and Tetrapyrrole Biosynthesis in Arabidopsis thaliana

“…LIL3 has been detected in protein fractions of etiochloroplasts of barley (Hordeum vulgare), which also contain protochlorophyll (Reisinger et al, 2008). LIL3 proteins also are present in fractions of Arabidopsis chloroplasts that contain Chlide and geranylgeranyl pyrophosphate, which supports the findings mentioned above regarding LIL3's involvement in late steps of terpenoid and Chl biosynthesis (Mork-Jansson et al, 2015a). Finally, the association of LIL3 with Chl has been demonstrated directly using a thermophoresis approach (MorkJansson et al, 2015b).…”

“…It remains a challenging task to assign further functions to LIL3, apart from its contribution to protein stability in Chl biosynthesis (Tanaka et al, 2010) and the assembly of proteins into the photosynthetic complexes (Lohscheider et al, 2015;Mork-Jansson et al, 2015a). Functions of LIL3 in Chl and precursor transfer require at least the physical interaction of LIL3 with other proteins.…”

“…To assess the effects of a relative lack of LIL3 on the assembly and repair of photosynthetic complexes (Lohscheider et al, 2015;Mork-Jansson et al, 2015a), we performed BN-PAGE to examine photosystem-LHC complex formation. It was obvious that the amounts of stable supercomplexes are diminished as a result of LIL3 deficiency (Fig.…”

“…1) is followed by the two transmembrane domains and a hydrophilic C-terminal segment that protrudes into the stroma of the plastid. In previous reports (Tanaka et al, 2010;Takahashi et al, 2014;Mork-Jansson et al, 2015a, 2015b, LIL3 was proposed to serve as a membrane anchor for other proteins and as a binding site for Chl. Specifically, it was proposed that LIL3 contributes to the formation of diverse protein complexes involved in Chl synthesis or to the assembly of Chl into Chl-binding proteins during their integration into the thylakoid membrane and subsequent assembly into photosynthetic protein complexes.…”

“…The stability of macromolecular protein assemblies is facilitated through direct physical interactions among the proteins. This interplay is often supported by nonenzymatic chaperones and auxiliary factors (Tanaka et al, 2010;Albus et al, 2012;Mork-Jansson et al, 2015a).…”