Nuclear-Cytoplasmic Translocations of Protein p26 during Aerobic-Anoxic Transitions in Embryos of Artemia franciscana

Abstract

Embryos of the crustacean Artemia franciscana survive continuous anoxia for periods of years, during which their metabolism comes to a reversible stand-still. A question of some interest concerns the maintenance of cellular integrity in the absence of biosynthesis and an ongoing energy metabolism. The present paper continues previous work on an abundant protein (p26) that undergoes extensive intracellular translocation during aerobic-anoxic transitions, exhibits several characteristics of stress proteins, and might be involved in metabolic regulation during aerobic-anoxic transitions. Since it has been established that intracellular pH (pHi) plays a major role in aerobic-anoxic transitions in this system we examined the pH-dependence of nuclear-cytoplasmic translocations of p26. In unincubated and aerobic-incubated embryos (pHi ⩾ 7.9) p26 was located in the "soluble" fraction, whereas in anoxic embryos (pH about 6.3) roughly 50% was translocated into the nucleus as shown by immunocolloidal gold electron microscopy. These nuclear translocations also took place in vitro, simply by manipulating buffer pH in a physiologically appropriate fashion. Immunostaining of Western blots prepared after two-dimensional electrophoresis revealed several isoforms of native p26. The isoelectric point of the major isoform was 7.10 ± 0.05, a value close to the pH at which p26 translocation into the nucleus was first initiated in vitro. 31 P-NMR measurements indicated that pHi was maintained at acidic levels (about 6.3) during prolonged anoxia. We also found that pHi of hydrated (0°C) but otherwise unincubated embryos was alkaline, allowing for rapid resumption of metabolism under permissive conditions. The significance of these pH-dependent translocations of p26 is discussed.