Cryoprotectin binds to thylakoid membranes and reduces membrane fluidity

Hany Sror, Dirk K. Hincha und Jürgen M. Schmitt
Institut für Pflanzenphysiologie, Freie Universität Berlin, 14195 Berlin, Germany

It has been show that a protein (Cryoprotectin) from cold-acclimated cabbage (Brassica oleracea L.) protects thylakoid
membranes against freeze - thaw damage. Protein sequencing of purified cryoprotectin showed that it is a member of the lipid
transfer protein (LTP) gene family. The functional mechanism of cryoprotection is unknown. In this study the binding of
cryoprotectin with thylakoid membranes and the associated changes in the physical properties of the membranes were studied as
a possible mechanism of cryoprotection by cryoprotectin. The results show that cryoprotective activity can be removed from
cryoprotectin crude extract by thylakoid membranes. Antibodies raised against two synthetic peptides derived from a
non-specific lipid transfer protein (wax9) identified a 10KD protein in western blots of cryoprotectin crude extract. This protein
disappeared gradually from the western blots of supernatants and was found in the western blots of thylakoid pellet proteins.
Evidence for binding of cryoprotectin with thylakoids was obtained by determination of freeze - thaw damage of pre-loaded
thylakoids after washing for different times. The results show that cryoprotection does not change by washing of thylakoids
pre-loaded with cryoprotectin and also the 10KD protein does not disappear from the western blots of thylakoid pellets after
several rounds of washing. Presence of 5mM glucose 6-sulfate in the cryoprotectin crude extract completely inhibits the
cryoprotective activity as well as the binding of thylakoids with cryoprotectin as demonstrated by western blots of supernatants
and thylakoid pellets. Fluorescence depolarization measurements with both probes trimethyl ammonium -1,6diphenyl 1,3,5
hexatriene (TMA-DPH) and trimethyl ammonium propyl -1,6diphenyl 1,3,5 hexatriene (TMAP-DPH) showed a significant
reduction of lipid fluidity in the presence of cryoprotectin. In contrast the hydrophobic core of the membrane as probed with 1,6
diphenyl-1,3,5 hexatriene (DPH) was not influenced . While in the presence of 5mM glucose 6 sulfate the fluidity of thylakoid
membranes was not affected in both hydrophobic region probed by DPH and the hydrophilic part and head group region
probed by TMA-DPH and TMAP-DPH .

The data strongly suggest that the mechanism of cryoprotection includes binding with thylakoids and reduction of the fluidity
of membrane lipids in the hydrophilic region, resulting in a lower solute permeability of the membranes and preventing freeze -
induced solute loading.

presented at: XI. Havel-Spree-Kolloquium, 2.12.2000, Humboldt-Universität, Berlin.