Light- and Ca2+-Modulated Heterotrimeric GTPases in the Eyespot Apparatus of a Flagellate Green Alga

Abstract

Little is known about phototactic signal transduction in flagellate green algae; therefore, eyespot apparatuses, which are the light-sensitive “organelles” involved in photoorientation of these algae, were isolated and analyzed for the presence of heterotrimeric guanine nucleotide binding proteins (G proteins) and their coupling to the retinal-based photoreceptor. Specific high-affinity ³⁵S-GTP-γ-S binding and GTPase activity, with sensitivity toward antibodies raised against vertebrate/invertebrate G_α subunits and fluoroaluminates, were detected. In one- and two-dimensional immunoblot analyses, an antiserum directed against G_iα-type subunits exhibited cross-reactivity at 42 kD, whereas a 43-kD protein cross-reacted with antisera directed against G_qα subunits. Green light below 1 μE m⁻² sec⁻¹ suppressed cholera toxin–dependent ADP ribosylation at these apparent molecular masses and modulated a significant proportion of the GTPase activity in a reversible manner. Antisera against Chlamydomonas rhodopsin and the G_α subunits completely impaired light modulation. Both light sensitivity and dark recovery of the GTPase were affected by changes in free Ca²⁺. Dissociation of the putative G_α subunits from the eyespot membranes was not observed when the membranes were illuminated. Our results emphasize the regulatory potential of G_α subunits in rhodopsin-based signaling of flagellate green algae.