Abstract
Chloroplast biogenesis involves the coordinated expression of the plastid and nuclear genomes, requiring information to be sent from the nucleus to the developing chloroplasts and vice versa. Although it is well known how the nucleus controls chloroplast development, it is still poorly understood how the plastid communicates with the nucleus. Currently, haem is proposed as a plastid-to-nucleus (retrograde) signal that is involved in various physiological regulations, such as photosynthesis-associated nuclear genes expression and cell cycle in plants and algae. However, components that transduce haem-dependent signalling are still unidentified. In this study, by using haem-immobilized high-performance affinity beads, we performed proteomic analysis of haem-binding proteins from Arabidopsis thaliana and Cyanidioschyzon merolae. Most of the identified proteins were non-canonical haemoproteins localized in various organelles. Interestingly, half of the identified proteins were nucleus proteins, some of them have a similar function or localization in either or both organisms. Following biochemical analysis of selective proteins demonstrated haem binding. This study firstly demonstrates that nucleus proteins in plant and algae show haem-binding properties.This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.
Highlights
Haem serve as cofactors of haemoproteins in various organelles that function in mitochondria respiratory and chloroplast photosynthetic electron transport chains, and in the detoxification of reactive oxygen species and xenobiotics, as well as in oxygen storage and transport [1]
Concerning haem regulation, we recently found that GUN1, which is a pentatricopeptide repeat (PPR) protein with a small MutS-related (SMR) domain and is the central integrator of retrograde signalling [34], binds to haem and modulates tetrapyrrole biosynthesis [35]
Canonical haemoproteins were not involved in haemin-binding proteins, probably covalently or noncovalently attached haem prevented the binding to proteins
Summary
Haem serve as cofactors of haemoproteins in various organelles that function in mitochondria respiratory and chloroplast photosynthetic electron transport chains, and in the detoxification of reactive oxygen species and xenobiotics, as well as in oxygen storage and transport [1]. In the green algae Chlamydomonas reinhardtii, a single FC encodes a plastid-localized FC protein [9], while in the red algae Cyanidioschyzon merolae, FC is only found in mitochondrial extracts [10] These results suggest that in Streptophyta and Cholorphyta, the dominant plastid FC activity supplies haem for the plastid as well as other organelle-localized haemoproteins, while distinct mitochondrial haem biosynthesis is employed in Rhodophyta. To elucidate the molecular mechanism of haem trafficking and signalling role, it is important to identify its molecular target(s) For this purpose, we have developed haemimmobilized high-performance affinity beads that allow single-step affinity purification of drug target proteins from crude cell extracts [26]. We performed affinity purification of haem-binding proteins from A. thaliana and Cy. melorae cell extracts. After incubation for 4 h, the cell extracts were used for the affinity purification assay using haemin-agarose beads and analysed by western blot analysis using anti-His-tag antibodies. The SRT1 activity assay was performed as described in the manufacturer’s protocol of the SIRT-Glo assay (Promega) using 0.3 μM of purified SRT1
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