Hendershot L M, Wei J Y, Gaut J R, Lawson B, Freiden P J, Murti K G
Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Mol Biol Cell. 1995 Mar;6(3):283-96. doi: 10.1091/mbc.6.3.283.
BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.
BiP具有ATP结合/水解活性，人们认为这对于其在内质网（ER）中陪伴蛋白质折叠和组装的能力至关重要。我们在仓鼠BiP克隆中产生了一系列抑制ATP酶活性的点突变，并生成了一种物种特异性抗BiP抗体，以监测突变仓鼠BiP在COS猴细胞中表达的影响。BiP的酶失活在体内并不干扰其与Ig重链结合的能力，但在体外确实抑制了ATP介导的重链释放。免疫荧光染色和电子显微镜显示，表达BiP ATP酶突变体的COS细胞中内质网出现囊泡化。当不包含蛋白质结合结构域的BiP“44K”片段发生类似突变时，未观察到内质网破坏，但当BiP的蛋白质结合区域在没有ATP结合结构域的情况下表达时，则观察到内质网破坏。这表明作为无活性伴侣蛋白的BiP与靶蛋白的结合是内质网破坏的原因。这是关于哺乳动物BiP突变体体内表达的首次报道，证明体外鉴定的ATP酶突变体在体内表达时表现为显性负突变体。
