Isolation and characterization of a G protein alpha subunit gene from Stentor coeruleus

Abstract

Stentor coeruleus is a unicelluar heterotrich ciliate that can exhibit a step-up photophobic response and a negative phototactic response. The photoreceptor, stentorin, is located in the pigment granule just below the cell membrane. Proton transfer from the excited state stentorin is thought to be an initial process and subsequent calcium influx induces the ciliary beat reversal. Recent evidence suggests that cGMP and heterotrimeric G proteins are involved in this light signal transduction pathway as in the vertebrate visual system. Furthermore, a 39 kDa protein from the Stentor membrane fraction was detected by immunoblot analysis with the AS/7 antisera against the carboxyl decapeptide of transducin $\alpha$ subunit. As a first step to trace each molecule in the light signal transduction pathway, a G protein $\alpha$ subunit gene from Stentor was cloned by the PCR approach. Even though just part of the G$\alpha$ subunit gene was cloned, it is homologous to the mammalian Gi type $\alpha$ subunit (amino acid sequence identity is 60%). To check the possibility that stentorin may interact with a G protein directly, the location of the G protein $\alpha$ subunit was investigated by immunogold labeling of the AS/7 antisera. The result indicated that the G protein is located in the myoneme and the kinetosome fibre. These organelles are responsible for cell contraction and cell extension