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(α)q subunits. Green light below I μEm-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 Gα 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 Gα subunits from the eyespot membranes was not observed when the membranes were illuminated. Our results emphasize the regulatory potential of G. subunits in rhodopsin-based signaling of flagellate green algae.