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.