The accumulation of triacylglycerols within the endoplasmic reticulum of developing seeds of Helianthus annuus

Microsomes isolated from developing seeds of Helianthus annuus were prepare d in a medium which ensured that endoplasmic reticulum (ER)-bound polysomes remained attached to the ER during homogenization. The microsomes were the n incubated with the substrates necessary to sustain the synthesis of triac ylglycerols (TAGs). Microsomes that contained high activities of the enzyme s involved in the synthesis of TAGs (the enzymes of the Kennedy pathway) ac cumulated TAGs synthesized in vitro, resulting in a decrease in their buoya nt density. These light membrane fractions could therefore be separated on discontinuous sucrose density gradients from microsomes containing low acti vities of the enzymes of the Kennedy pathway. Analysis of the microsome fra ctions by H-1-NMR spectroscopy showed that the TAGs synthesized in the micr osomes in vitro were tumbling isotropically in an environment similar to th at of the TAGs in oil bodies. Western blot analysis revealed that microsome s which synthesized large amounts of TAGs in vitro were also substantially enriched in oleosins. In addition, labelling studies indicated that the ole osins newly synthesized in vitro by 'run-on' translation of ER-bound polyso mes also localized to light membrane fractions. This indicates that oleosin s are specifically enriched in regions of the ER involved in the biogenesis of the oil body.