Analysis of the BiP gene and identification of an ER retention signal in Schizosaccharomyces pombe.
Analysis of the BiP gene and identification of an ER retention signal in Schizosaccharomyces pombe.
复制标题
粟酒裂殖酵母 BiP 基因的分析和 ER 滞留信号的鉴定。
作者：
Alison L. Pidoux;John Armstrong
我们从分裂酵母Schizosaccharomyces pombe中克隆了常驻腔内内质网蛋白BiP的基因。预测的蛋白质产物同样不同于芽殖酵母和哺乳动物同源物。在S. pombe中，BiP基因的破坏是致命的，BiP mRNA水平受到包括热休克在内的各种应激的调节。表达表位标记的BiP蛋白的细胞的免疫荧光显示它定位于核包膜，围绕细胞周围，并通过细胞质形成网状结构。出乎意料的是，我们发现BiP蛋白含有一个可以利用的N -连锁糖基化位点。BiP的C端四个氨基酸是Ala - Asp - Glu - Leu，这是在腔内质网蛋白C端发现的XDEL序列的新变体。为了确定该序列是否在S.pombe中作为分选信号，我们表达了一个酸性磷酸酶融合蛋白，该蛋白在其C端与氨基酸ADEL延伸。对该融合蛋白的分选分析表明，ADEL序列足以引起蛋白质在内质网中的保留。DDEL、HDEL和KDEL序列也可以直接影响pombe酸性磷酸酶的ER -滞留。
We have cloned the gene for the resident luminal ER protein BiP from the fission yeast, Schizosaccharomyces pombe. The predicted protein product is equally divergent from the budding yeast and mammalian homologues. Disruption of the BiP gene in S. pombe is lethal and BiP mRNA levels are regulated by a variety of stresses including heat shock. Immunofluorescence of cells expressing an epitope‐tagged BiP protein show it to be localized to the nuclear envelope, around the cell periphery and in a reticular structure through the cytoplasm. Unexpectedly, we find the BiP protein contains an N‐linked glycosylation site which can be utilized. The C‐terminal four amino acids of BiP are Ala‐Asp‐Glu‐Leu, a new variant of the XDEL sequence found at the C‐termini of luminal endoplasmic reticulum proteins. To determine whether this sequence acts as a sorting signal in S.pombe we expressed an acid phosphatase fusion protein extended at its C‐terminus with the amino acids ADEL. Analysis of the sorting of this fusion protein indicates that the ADEL sequence is sufficient to cause the retention of proteins in the endoplasmic reticulum. The sequences DDEL, HDEL and KDEL can also direct ER‐retention of acid phosphatase in S.pombe.