74.3 Friday, Jan. 6 Late Embryogenesis Abundant Protein Ameliorates Inhibition of Mitochondrial Respiration MARUNDE, M.R.*; LI, S.; HAND, S.C.; MENZE, M.A.; Dept. Biol. Sci., Eastern Illinois University, Charleston, IL; Dept. Biol. Sci., Louisiana State University, Baton Rouge, LA; Dept. Biol. Sci., Louisiana State University, Baton Rouge, LA; Dept. Biol. Sci., Eastern Illinois University, Charleston, IL firstname.lastname@example.org
Mechanisms that govern anhydrobiosis involve the accumulation of highly hydrophilic macromolecules, such as late embryogenesis abundant (LEA) proteins. Warner et al. (2009) reported group 1 LEA proteins in Artemia franciscana embryos, which were localized within the cytoplasm or the mitochondrion. We designed primers based on NCBI sequence ACX81198 to amplify cDNA from A. franciscana. Two variants encoding for proteins of 181 (LEA1.1) and 197 (LEA1.3) amino-acid lengths were cloned and expressed in Drosophila melanogaster cells (Kc167). Confocal microscopy revealed a construct composed of green fluorescence protein (GFP) and LEA1.3 accumulates in the mitochondria (LEA1.3-GFP), while LEA1.1-GFP was predominantly found in the cytoplasm. No significant difference in routine respiration was observed among Kc167 controls and cell lines that stably expressed LEA1.3, LEA1.3-GFP, or LEA1.1-GFP (n = 9). Routine respiration of intact Kc167 cells at 25 °C was 17.7 ± 1.3 pmol O2•s-1•10-6 cells (n = 18, ±SD). Acute exposure to 500 mM NaCl reduces respiration in controls by 46 ± 4% (n = 6, ±SD) and expression of LEA1.3 did not ameliorate this inhibition. In presence of mixed substrates oxygen consumption of permeabilized control cells was 16.8 ± 2.8 pmol O2•s-1•10-6 cells, and addition of 2 mM ADP increased oxygen flux by 1.7 ± 0.21-fold (n = 14, ±SD). Titrations of KCl up to 500 mM led to successive drops in oxygen flux. This inhibition was significantly ameliorated in Kc167-LEA1.3 cells. During desiccation intracellular ion concentrations will increase. Our results suggest that LEA1.3 exerts a protective influence on mitochondrial function (NSF-IOS-0920254, CFR-EIU-2010).