G proteins and Ca²⁺-modulated protein kinases of a plasma membrane-enriched fraction and isolated eyespot apparatuses ofSpermatozopsis similis(chlorophyceae)

Abstract

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: (i) the isolation and characterisation of an enriched plasma membrane fraction of Spermatozopsis similis, and (ii) a comparison of the distribution of Ca2+-dependent putative kinases and G proteins, as recognised by antibodies directed against mammalian Gc~-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 c~-subunits (anti-c~ common). A 43 kDa protein was recognised by a serum against the G~-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(~-subunits also labelled a 43 kDa and 54 kDa protein in the eyespot preparations. Additionally, four putative Ca2+-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 were also enriched in the eyespot fraction but not in other subcellutar fractions. Monoclonal antibodies directed against a soybean CaZ+-dependent protein kinase cross-reacted with a 6I 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.

The Phosphoproteome of aChlamydomonas reinhardtiiEyespot Fraction Includes Key Proteins of the Light Signaling Pathway

“…However, in an extended eyespot proteome analysis, two kinases of this type have been detected recently (M. Mittag and G. Kreimer, unpublished data). Their calculated molecular masses (67 and 54 kD) are similar to proteins exhibiting Ca 21 -dependent autophosphorylation in eyespot preparations of S. similis (77, 48, and 47 kD; Linden and Kreimer, 1995;Schlicher et al, 1995). Due to the outlined central role of Ca 21 in eyespotrelated signaling, it seems likely that the identified Ca 21 -binding phosphoprotein might be one member of this light-driven signaling cascade.…”

“…Lipid removal, protein precipitation, and SDS-PAGE were conducted as described (Kreimer et al, 1991;Calenberg et al, 1998). Western-blot analyses followed basically the protocol of Schlicher et al (1995) with the following modifications: Skim milk powder was replaced by bovine serum albumin, the first blocking step was reduced to 2 h, and incubations with the polyclonal antiphospho-Thr antibody and the monoclonal anti-phosphor-Tyr antibody (Cell Signaling Technologies; both used at a dilution of 1:1,000) were done overnight at 4°C. Images of blots were taken with a Coolpix 990 (Nikon) and processed with Photoshop (Adobe Systems).…”

Vision in microalgae

“…Membrane-associated G-protein a-subunits have been identi®ed in Chlamydomonas reinhardtii and Spermatozopsis similis (Hegemann and Harz 1993;Schlicher et al 1995). However, it has only been convincingly demonstrated in Spermatozopsis that the GTPase activity is light regulated with an action spectrum similar to that of rhodopsin.…”

Sensory Rhodopsin Signaling in Green Flagellate Algae

“…The CSRB-mediated current appears to be primarily generated by this second mechanism. Several enzymes characteristic of signal transduction pathways in animals, including heteromeric GTPases, have been detected in isolated eyespot preparations of green f lagellate algae (Schlicher et al, 1995;Linden and Kreimer, 1995;Calenberg et al, 1998), although whether they play a role in photomotility signaling has yet to be determined.…”