Holkeri H, Paunola E, Jämsä E, Makarow M
Institute of Biotechnology, University of Helsinki, Finland.
J Cell Sci. 1998 Mar;111 ( Pt 6):749-57. doi: 10.1242/jcs.111.6.749.
We used the rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli ss-lactamase to dissect the functions of Saccharomyces cerevisiae BiP/Kar2p in vivo. Both were fused to the Hsp150Delta-polypeptide, which promotes proper folding of heterologous proteins which otherwise are misfolded in the yeast ER. Hsp150Delta-NGFRe and Hsp150Delta-beta-lactamase acquired disulfides and were properly folded and ONcreted to the culture medium. When disulfide formation was prevented by incubating cells with dithiothreitol (DTT), Hsp150Delta-NGFRe remained in the endoplasmic reticulum (ER). The occupancy of an otherwise partially used N-glycosylation site of reduced NGFRe was complete suggesting that, normally, folding and disulfide formation occurred as rapidly as N-glycosylation. Removal of DTT resulted in remarkably rapid disulfide formation and secretion, suggesting only mild conformational distortion of reduced NGFRe. In contrast, reduced Hsp150(Delta)-ss-lactamase was severely misfolded and attained a secretion competent conformation more slowly after reoxidation. When kar2-159 cells were incubated at permissive temperature 24 degrees C with DTT, the reporter proteins were retained in the ER. After shift of the cells to 34 degrees C to inactivate BiP/Kar2p irreversibly, and subsequent removal of DTT, most pre-accumulated Hsp150Delta-NGFRe was rapidly secreted, whereas Hsp150Delta-beta-lactamase was secretion incompetent. Thus, Hsp150Delta-NGFRe did not require BiP/Kar2p for conformational maturation, though translocation was dependent on BiP/Kar2p. Apparently proteins differ in their post-translocational requirements for BiP/Kar2p, indicating that translocation and chaperoning are distinct functions.
我们利用大鼠神经生长因子受体胞外结构域（NGFRe）和大肠杆菌β-内酰胺酶在体内剖析酿酒酵母BiP/Kar2p的功能。二者均与Hsp150Delta-多肽融合，该多肽可促进异源蛋白正确折叠，否则这些异源蛋白在酵母内质网中会错误折叠。Hsp150Delta-NGFRe和Hsp150Delta-β-内酰胺酶形成了二硫键，正确折叠并分泌到培养基中。当用二硫苏糖醇（DTT）处理细胞以阻止二硫键形成时，Hsp150Delta-NGFRe保留在内质网（ER）中。还原型NGFRe原本部分使用的N-糖基化位点被完全占据，这表明正常情况下，折叠和二硫键形成与N-糖基化一样迅速。去除DTT后，二硫键形成和分泌非常迅速，这表明还原型NGFRe仅存在轻微的构象扭曲。相比之下，还原型Hsp150(Delta)-β-内酰胺酶严重错误折叠，再氧化后达到分泌能力构象的速度更慢。当kar2-159细胞在允许温度24℃下与DTT一起孵育时，报告蛋白保留在内质网中。将细胞转移至34℃以不可逆地使BiP/Kar2p失活，随后去除DTT后，大多数预先积累的Hsp150Delta-NGFRe迅速分泌，而Hsp150Delta-β-内酰胺酶则无分泌能力。因此，Hsp150Delta-NGFRe的构象成熟不需要BiP/Kar2p，尽管转运依赖于BiP/Kar2p。显然，蛋白质在转运后对BiP/Kar2p的需求不同，这表明转运和伴侣作用是不同的功能。
