Oktaedrische vakuoläre Proteinkristalle in Phycomyces blakesleeanus: Biochemische und fluoreszenzspektroskopische Charakterisierung

The negatively gravitropic sporangiophores of the zygomycete Phycomyces blakesleeanus contain vacuolar paracrystalline protein bodies. These so-called octahedral crystals sediment and passively relocate upon reorientation of the sporangiophore. Therefore, a function as statoliths in gravisusception is currently discussed. The specific density of isolated crystals varies between 1,247 and 1,271 g cm-3. SDS-PAGE of purified crystals reveals three major proteins with relative molecular masses of 16, 46,5 and 55 kDa. These proteins are absent in gravitropic mutants which lack the crystals. Absorption spectra of suspensions of isolated crystals indicated the presence of chromphores. Spectrofluorometric data confirmed the flavin- or pterin-like nature, respectively, of the chromophores associated with the octahedral crystals. Reduction or oxidation in vitro revealed redox-dependent binding of the flavin-like chromophores to the crystal. Riboflavin bound with a KD, ox of 6x10-5M and a KD, red of 2,8x10-5M, respectively. The binding capacity of a single average crystal (the average height of wild-type crystals was 3,91 µm, corresponding to a volume of 9,96 µm³) was estimated about 106 pigment molecules. While flavin-like chromophores bound in the reduced form to the crystals, the pterin-like pigments bound in the oxidized form. This redox-combination raises the possibility electron transfer between the non-covalently bound chromophores, possibly as a step in the signal transduction chain. Fluorescence polarization spectra showed that the chromophores are arranged almost in parallel within the protein matrix of the crystal. This finding and the large overlap of the fluorescence emission spectrum of the pterin-like pigment(s) with the excitation spectrum of the flavin-like chromophore may well allow intermolecular energy (Förster) transfer. Studies on gravitropic bending suggested that the statolith function of the crystals is less important for gravisusception than two other stimuli, wall strain and lipid globules. However, stimulation of gravitropic bending by tonic red light in the presence of crystals with supportive spectral properties of the associated chromophores suggests an important function of the crystals for integration of the phototropic and gravitropic signaling pathways. First results indicate a role of Ca2+ on the bending of crystal-carrying sporangiophores. Support for this comes from sequence homologies of the crystal matrix protein (55 kDa) to a cGMP-regulated channel involved in phototransduction, to a calcium binding protein and to a vacuolar ATPase. So far, no sequence homologies to already known flavoproteins could be detected. The function of the octahedral crystals of Phycomyces blaskesleeanus appears not restricted to statolith function but rather the crystals have to be considered as essential elements for the integration of the signal transduction chains of gravitropism and phototropism.