Biochemical characterization of the self-splicing GIN1 protein in arbuscular mycorrhizal fungi

Abstract

Arbuscular mycorrhizal fungi are obligate symbionts, which means they can not complete their life cycle without establishing symbiosis with a host plant. Nevertheless, mycorrhizal spores are able to germinate and develop a limited hyphal growth in search for a compatible host. In the absence of a host root signal, the fungus stops and arrests growth. When the fungus meets a compatible host root, the fungus forms appressoria on the root rhizodermis and constitutes a functional symbiosis. Because of its beneficial plant effects, it is of outstanding interest to investigate the mechanism of recognition and establishment of the symbiosis between the two partners. Fungal genes that are exclusively expressed during the pre-symbiotic or early developmental stages of the AM symbiosis have been described for the AM fungus Glomus mosseae (Requena et al., 2002). A novel gene called GmGIN1, coding for GTPase Intein, was identified. GmGIN1 encodes for a two-domain structure protein, with a putative self-splicing site in the junction of the two domains.

GmGIN1 carboxy terminus shares similarity with the carboxy terminus of Hedgehog (Hh) selfs-plicing proteins. This domain is responsible for selfsplicing of the protein precursor into two different peptides. In Hh proteins, the first nucleophilic attack is produced by an internal conserved cysteine, which is situated in GmGIN1 at the 237 amino acid position. The second nucleophilic attack is triggered by a cholesterol molecule, which is covalently attached to the processed N-terminus and determines its localization at raft domains in the cell membrane. In order to investigate an analogous processing mechanism of GmGIN1, the C-terminus was heterologously expressed in E. coli. Purified protein was shown to undergo selfsplicing in the same conditions as the carboxy terminus of Hh proteins. Nevertheless, yields of purified protein were not sufficient to appreciate the splicing induction by addition of cholesterol. Lipid extracts from mycorrhizal samples of the closely related fungus G. intraradices were performed in order to investigate which molecule could be responsible for the second nucleophilic attack in vivo. Mycorrhizal root sterol extracts were able to induce selfsplicing of the carboxy terminus of D. melanogaster Hh, expressed and purified under the same conditions as GmGIN1-C. Lipid analysis confirmed the presence of cholesterol derivatives, and traces of cholesterol itself, in the extracts. Expression of the full length GmGIN1 protein was achieved, but the amount of active purified protein was not enough to observe the self-splicing activity.

The amino terminal domain of GmGIN1 shares homology to a new family of GTPases, the AIG/GIMAP family, conserved among plants, fungi and animals. GmGIN1-N contains three of the five conserved cassettes implicated in GTP binding and hydrolysis. Two of these motifs are also involved in binding to other nucleotide derivatives, such as ATP. In order to investigate the nucleotide binding and possible hydrolysis activity of GmGIN1-N, this domain was heterologously expressed in E. coli. No selective binding to á-32P-GTP was observed in overlay assays, neither for GmGIN1-N nor for the positive controls Umcdc42 wild type and Umcdc42(Q-L) mutant. Optimal binding was observed for the positive controls purified under native conditions after incubation with 35S-ã-GTP in solution. GmGIN1-N was shown also to bind to 35S-ã-GTP, but to a lower extent. Competition analysis by 35S-ã-GTP dissociation assays demonstrated that GmGIN1-N has a higher affinity for ATP compared to GTP. This was confirmed by the nucleotide hydrolysis assays, in which GmGIN1-N was shown to actively hydrolyze ATP, but not GTP. This protein could be involved either in recognition events and establishment of the symbiosis upon meeting a host plant, or in growth arrest and programmed cell death in the absence of a compatible partner.

Arbuskuläre Mykorrhizapilze sind obligate Symbionten. Ihre Entwicklung ist abhängig von einer Wirtspflanze. Das GmGIN1 Protein spielt vermutlich bei der Erkennung zwischen Wirtspflanze und symbiontischem Pilz eine Rolle oder es ist beteiligt am Wachstumsstop sowie programmierten Zelltot in Abwesenheit eines geeigneten Partners. GmGIN1 kodiert für ein Protein mit zwei Domänen. Diese sind durch eine selbst-spleissende Stelle verbunden. Der N-Terminus von GmGIN1 ähnelt einer GTPase, der C-Terminus ähnelt dem C-Terminus von Hedgehogproteinen. In dieser Arbeit wurde die GTPase Aktivität und die Selbst-Spleissungseigenschaften von GmGIN1 untersucht.