Association of DNA polymerase alpha and beta with rough endoplasmic reticulum in sea-urchin eggs and changes in subcellular distribution during early embryogenesis

Abstract

The subcellular distribution of DNA polymerase alpha and beta was examined in unfertilized eggs and embryos of the sea-urchin, Hemicentrotus pulcherrimus. In unfertilized egg homogenates, prepared in sucrose solution containing 5 mM 2-mercaptoethanol and 5 mM MgCl2, DNA polymerases equilibrated in isospycnic centrifugation as a single peak at a buoyant density of 1.261 g/cm3 (band I). This indicates that DNA polymerases associate with a cytoplasmic organelle. Band I was converted to bands II (1.227 g/cm3), III (1.177 g/cm3) and IV (1.146 g/cm3) by EDTA treatment. The RNA content of bands I, II, III and IV was approximately 0.34, 0.25, 0.10 and 0.04 mg/mg protein respectively. In isokinetic and isopycnic centrifugations both DNA polymerase and RNA cosedimented and coequilibrated. These results suggest that bands I, II, III and IV contain various amounts of ribosomes on a common structure. Examination by electron microscopy indicated that bands I, II, III and IV contained mainly monolayered membrane vesicles with different amounts of bound ribosomes. The content of ribosomes varied in the order: band I > band II > band III > band IV. Each band contained DNA polymerase with sedimentation coefficients of 5.8-7.6 S (sensitive to N-ethylmaleimide and aphidicolin) and 3.2 S (insensitive to these drugs). We conclude that almost all of DNA polymerases alpha and beta are localized on the rough endoplasmic reticulum in unfertilized eggs. Mixing experiments suggest that the association is specific and is not an artifact of homogenization. During development of embryos DNA polymerases associated with the membrane decreased with concomitant increase in the nuclear fraction. This suggests that the enzymes migrate from the cytoplasm to the nucleus. It is discussed that the role of the endoplasmic reticulum as a storage site of DNA polymerases in unfertilized eggs and the mechanism of translocation of DNA polymerases from the cytoplasm to the nucleus during early embryogenesis.