Chao Q, Etzler M E
Section of Molecular and Cellular Biology, University of California, Davis 95616.
J Biol Chem. 1994 Aug 19;269(33):20866-71.
Two soluble vacuolar lectins, seed lectin and DB58, from the legume, Dolichos biflorus, were expressed in Saccharomyces cerevisiae using both low and high copy number plasmids under the control of the GAL1 promoter. When expressed at low levels, these lectins were each secreted by the yeast at all stages of growth. Expression of the lectins at high levels resulted in the retention of most of the lectins in the cell. Cell fractionation studies showed that this retained lectin was not associated with the yeast vacuoles. The differential COOH-terminal processing of these lectins, that results in the production of heteroligomers in plants, did not occur in yeast. Site-directed mutagenesis was employed to produce a construct encoding the shorter subunit of the seed lectin. Expression of this truncated subunit in yeast produced the same results as found with the larger subunit, thus indicating that this modification does not provide the vacuolar targeting signal. The inability of these two vacuolar proteins from different plant tissues to be transported to the yeast vacuole suggests that plants and yeast utilize different signals for targeting soluble vacuolar proteins.
利用低拷贝数和高拷贝数质粒，在GAL1启动子的控制下，将豆科植物双花扁豆的两种可溶性液泡凝集素（种子凝集素和DB58）在酿酒酵母中表达。当低水平表达时，这些凝集素在酵母生长的各个阶段均由酵母分泌。高水平表达凝集素导致大多数凝集素保留在细胞内。细胞分级分离研究表明，这种保留的凝集素与酵母液泡无关。这些凝集素在植物中产生异源寡聚体的COOH末端差异加工在酵母中未发生。采用定点诱变产生编码种子凝集素较短亚基的构建体。该截短亚基在酵母中的表达产生了与较大亚基相同的结果，因此表明这种修饰并未提供液泡靶向信号。来自不同植物组织的这两种液泡蛋白无法转运至酵母液泡，这表明植物和酵母利用不同的信号来靶向可溶性液泡蛋白。
