G proteins and Ca super(2+)-modulated protein kinases of a plasma membrane-enriched fraction and isolated eyespot apparatuses of Spermatozopsis similis (Chlorophyceae)

• Published in: European journal of phycology Vol. 30; no. 4; pp. 319 - 330
• Main Authors: Schlicher, U, Linden, L, Calenberg, M, Kreimer, G
• Format: Journal Article
• Language: English
• Published: 01.01.1995
• Subjects: Chlorophyta; Spermatozopsis similis
• ISSN: 0967-0262

Knowledge about possible elements involved in signal transduction and adaptational phenomena during phototaxis of flagellate green algae is limited. In order to identify such putative elements we present here: (1) the isolation and characterisation of an enriched plasma membrane fraction of Spermatozopsis similis, and (ii) a comparison of the distribution of Ca super(2+)-dependent putative kinases and G proteins, as recognised by antibodies directed against mammalian G sub( alpha )-subunits, in the plasma membrane preparation and isolated eyespot apparatuses of this green alga. In western blot analyses a 54 kDa protein reacted with an antiserum against a conserved sequence of G protein alpha -subunits (anti- alpha common). A 43 kDa protein was recognised by a serum against the G sub( alpha )-subunit of transducin. These proteins were enriched in the eyespot fraction and to a lesser extent in the plasma membrane fraction. Two polyclonal antisera against C-terminal peptides of animal G sub( alpha )-subunit also labelled a 43 kDa protein in the eyespot preparations. Additionally, four putative Ca super(2+)-dependent kinases or their catalytic subunits (47, 48, 77 and 82 kDa) were identified in the plasma membrane fraction by a gel kinase assay following electrophoretic separation. The 82 kDa protein was specifically enriched in the plasma membrane fraction, while the other putative kinases wee also enriched in the eyespot fraction but not in other subcellular fractions. Monoclonal antibodies directed against a soybean Ca super(2+)-dependent protein kinase cross-reacted with a 61 kDa protein in the plasma membrane and a 60 kDa protein in the soluble fraction. Only a very faint response was observed in the fraction enriched in eyespot apparatuses. Our results indicate (i) a low cross-contamination of the eyespot apparatus preparation by normal plasma membrane constituents and (ii) the regulatory potential for the identified kinases and G proteins in blue/green-light-dependent photoresponses of Spermatozopsis similis.