Chlorophyll binding to light-harvesting-like protein 3

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. A function in tetrapyrrol synthesis and stabilization of geranylgeraniol reductase has been concluded from Lil3 mutants in Arabidopsis thaliana L. We show here that Lil3 binds Chlorophyll, and directly interacts with Chlorophyll synthase and Protochlorophyllide Oxidoreductase. Data indicate that Lil3 participates in the regulation of Chlorophyll biosynthesis. In etioplasts from barley fluorescent protein bands (F1, F2, and F3) assembled that all contained the light harvesting like protein Lil3 in a native PAGE approach when the plastids were supplemented with chlorophyllide and geranylgeraniol-pyrophosphate. Chlorophyllide triggered assembly of Lil3 in a low molecular weight band termed F3 containing protochlorophyllide-oxidoreductase and chlorophyll synthase. Upon phototransformation, chlorophyllide and chlorophyll esterified to geranylgeraniol accumulated in the band. Geranylgeraniolpyrophosphate triggered the increase of two higher molecular weight bands termed F1 and F2 and the decrease of band F3. F1 and F2 accumulated chlorophyll esterified to phytol. A more than 6-fold increase was determined of geranylgeraniolreductase in band F1 and of psb29, and D2 protein in band F2. Data indicated that Lil3 assembles in complex containing chlorophyll synthase, protochlorophyllide-oxidoreductase, and geranylgeraniolreductase. Direct interaction of Lil3 and between Lil3 and chlorophyll synthase, and protochlorophyllide oxidoreductase was identified in a split ubiquitin based yeast-two-hybrid analysis. However, no direct interaction with geranylgeraniolreductase was found. Thermophoretic analysis showed that Lil3.2 formed heterodimers with Lil3.1 (25 ± 7.8 nM) and that heterodimerization was favored relative to homodimerization (431 ± 59 nM). Lil3.2 was found to directly interact with chlorophyll a (231 ± 49 nM). Analysis indicated that heterodimerization of Lil3 reconstitutes a LHC chlorophyll binding motif, and is a prerequisite for binding of chlorophyll in Arabidopsis thaliana. Mork-Jansson AE, Gargano D, Kmiec K, Furnes C, Shevela D, Eichacker LA. (2015) Lil3 dimerization and chlorophyll binding in Arabidopsis thaliana. FEBS Lett., S0014-5793(15)00723-1. [Epub ahead of print]. Mork-Jansson A, Bue AK, Gargano D, Furnes C, Reisinger V, Arnold J, Kmiec K, Eichacker LA. (2015) Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley. PLoS One. 10, e0133145.