Loss of membrane proteins from thylakoids during freezing

Abstract

Slow freezing of thylakoids (about 0.5 K/min) in media containing an excess of cryotoxic solutes results in the release of membrane proteins and in the rapid inactivation of photophosphorylation and other processes dependent on structural intactness of membranes. Although loss of photophosphorylation was observed before protein release became extensive, both processes were dependent on the ratio of cryotoxic to cryoprotective solutes in the membrane suspensions. Moreover, inactivation of photophosphorylation and protein release during freezing were similarly influenced by different cryotoxic solutes. Cryotoxicity of different anions increased with increasing Stoke's law hydrated anion radius ( I > Br > Cl > F ). Divalent cations were more cryotoxic than monovalent alkali cations. Cryotoxicity thus followed a Hofmeister lyotropic power series. Two opposing effects of low temperature influenced protein release. Lowering the temperature increased accumulation of cryotoxic solutes in the membrane vicinity which promoted protein dissociation. However, rates of protein dissociation increased with increasing temperature. While the freezing temperature unequivocally determined the concentration of solutes in a solution coexisting with ice, both the extent of membrane inactivation and protein release depended on the initial concentration of solutes and on membrane concentration. Apparently, the volumes of the unfrozen hydrophilic phase and of the membranes are also important factors in freezing injury. The polypeptide pattern of released proteins differed drastically from that of thylakoids, but the same main proteins were released during freezing of membranes in the presence of different cryotoxic solutes. More than 35 polypeptide bands were observed in SDS-polyacrylamide gel electrophoretograms of released proteins. While the amount of protein released differed depending on freezing conditions and the composition of the suspending medium, more than 10% of the total membrane protein could be solubilized during freezing in the presence of sodium bromide or sodium iodide. Protein release during freezing is thought to be caused primarily by a suppression of intramembrane electrostatic interactions and ion competition although solute effects on water structure may also play a role.