Tague B W, Chrispeels M J
Department of Biology, University of California, San Diego, La Jolla 92093.
J Cell Biol. 1987 Nov;105(5):1971-9. doi: 10.1083/jcb.105.5.1971.
Phytohemagglutinin (PHA), the major seed lectin of the common bean, Phaseolus vulgaris, accumulates in the parenchyma cells of the cotyledons. It has been previously shown that PHA is cotranslationally inserted into the endoplasmic reticulum with cleavage of the NH2-terminal signal peptide. Two N-linked oligosaccharide side chains are added, one of which is modified to a complex type in the Golgi apparatus. PHA is then deposited in membrane-bound protein storage vacuoles which are biochemically and functionally equivalent to the vacuoles of yeast cells and the lysosomes of animal cells. We wished to determine whether yeast cells would recognize the vacuolar sorting determinant of PHA and target the protein to the yeast vacuole. We have expressed the gene for leukoagglutinating PHA (PHA-L) in yeast under control of the yeast acid phosphatase (PHO5) promoter. Under control of this promoter, PHA-L accumulates to 0.1% of the total yeast protein. PHA-L produced in yeast is glycosylated as expected for a yeast vacuolar glycoprotein. Cell fractionation studies show that PHA-L is efficiently transported to the yeast vacuole. This is the first demonstration that vacuolar targeting information is recognized between two highly divergent species. A small proportion of yeast PHA-L is secreted which may be due to inefficient recognition of the vacuolar sorting signal because of the presence of an uncleaved signal peptide on a subset of the PHA-L polypeptides. This system can now be used to identify the vacuolar sorting determinant of a plant vacuolar protein.
植物血凝素（PHA）是菜豆（Phaseolus vulgaris）的主要种子凝集素，它积累在子叶的薄壁细胞中。先前已经表明，PHA在共翻译过程中插入内质网，同时氨基末端信号肽被切割。添加了两条N-连接寡糖侧链，其中一条在高尔基体中被修饰为复合型。然后PHA被沉积在膜结合的蛋白质储存液泡中，这些液泡在生化和功能上与酵母细胞的液泡以及动物细胞的溶酶体相当。我们希望确定酵母细胞是否会识别PHA的液泡分选决定簇，并将该蛋白质靶向酵母液泡。我们在酵母酸性磷酸酶（PHO5）启动子的控制下，在酵母中表达了白细胞凝集PHA（PHA-L）的基因。在该启动子的控制下，PHA-L积累至酵母总蛋白的0.1%。酵母中产生的PHA-L如预期的那样被糖基化，成为酵母液泡糖蛋白。细胞分级分离研究表明，PHA-L被有效地转运到酵母液泡中。这是首次证明在两个高度不同的物种之间，液泡靶向信息能够被识别。一小部分酵母PHA-L会被分泌，这可能是由于一部分PHA-L多肽上存在未切割的信号肽，导致对液泡分选信号的识别效率低下。这个系统现在可用于鉴定植物液泡蛋白的液泡分选决定簇。
