Planta

, Volume 213, Issue 1, pp 51–63 | Cite as

Subfractionation of eyespot apparatuses from the green alga Spermatozopsis similis: isolation and characterization of eyespot globules

  • S. Renninger
  • E. Backendorf
  • G. Kreimer

Abstract.

Despite the well-characterized function of the green-algal eyespot apparatus as a combined absorption/reflection screen for the photoreceptor for phototaxis, little is known about the proteins involved in the formation of this complex organelle. We therefore purified the carotenoid-rich lipid globules, which are the most conspicuous component of the eyespot sensu strictu from Spermatozopsis similis Preisig et Melkonian. Electron microscopy and an average carotenoid:chlorophyll ratio of 51, confirmed the high purity of the fraction. The diameter of isolated globules (approx. 112 nm) fell within their in vivo range (90–120 nm). Absorption spectra in aqueous media peaked at 535 nm. The predominant carotenoids were β,ψ-, β, β- and δ-carotene. Freeze-fracture studies with cells and whole-mount electron microscopy of isolated globules demonstrated regularly arranged particles at the globule surface. Sodium dodecyl sulfate–polyacrylamide gel electrophresis revealed specific enrichment of 10 tightly bound major proteins and several minor proteins with the globules. Proteases were used to analyze their topology and function. Upon treatment with thermolysin, globules were released from a fraction enriched in isolated eyespot apparatuses. Major proteins of these globules, and those treated with thermolysin after isolation, were identical. However, the purified proteins were sensitive to thermolysin, indicating that domains of them are normally hidden in the globule matrix. In contrast, pronase degraded all globule-associated proteins in situ. These globules were not stable and easily fused, whereas thermolysin-treated globules were relatively stable. Lipase did not affect globule stability. These results indicate that the five thermolysin-resistant proteins (apparent Mr values: 56, 52, 32, 29, 27 kDa) are close to the surface and might be crucial for globule stabilization, whereas the thermolysin-accessible proteins are probably involved in globule/globule interactions and/or globule/eyespot-membrane interactions.

Key words: Eyespot apparatus Carotenoids Green alga Phototaxis Plastoglobule Spermatozopsis (eyespot) 

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Copyright information

© Springer-Verlag Berlin Heidelberg 2001

Authors and Affiliations

  • S. Renninger
    • 1
  • E. Backendorf
    • 1
  • G. Kreimer
    • 1
  1. 1.Botanisches Institut, Universität zu Köln, Gyrhofstrasse 15, 50931 Cologne, GermanyDE

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