Is glycosylation of aquaporins important for their membrane localization in gray treefrogs?

Abstract

In freeze‐tolerant Hyla chrysoscelis, cold acclimation and freezing involve accumulation of glycerol and distribution of glycerol and water among tissues and body fluids. Aquaporins, including HC‐1 (a water‐permeant protein) and HC‐3 (a glyceroporin conferring both water and glycerol permeability), facilitate those processes. These proteins are up‐regulated during cold acclimation, and glycosylated forms of the proteins are prominent. We hypothesized that glycosylation contributes to localization of the proteins in the plasma membrane. We tested this by fractionating liver tissue and examining the extent of glycosylation of aquaporins in membrane and cytosolic fractions, assessed by densitometry of Western blots with and without N‐glycosidase treatment of proteins. Specifics of glycosylation (banding patterns in Western blots) differed for membrane vs. cytoplasm, and for warm vs. cold. Preliminary studies suggest that, for HC‐1, expression in membrane was 3X that in cytoplasm in both warm and cold animals. Percent glycosylated HC‐1 was higher in membrane than cytoplasm, and in both fractions was reduced in cold acclimated animals. HC‐3 was about equally localized in membrane and cytoplasm in both warm and cold animals. We are as yet unable to detect an effect of acclimation on % glycosylation of HC‐3 (35–50% in both fractions, during both warm and cold acclimation). Supported by NSF IOB‐0517301.