Membrane Orientation of the Human Papillomavirus Type 16 E5 Oncoprotein
Membrane Orientation of the Human Papillomavirus Type 16 E5 Oncoprotein
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DOI：
10.1128/jvi.01968-09
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发表时间：
2010-02-15
影响因子：
5.4
通讯作者：
Schlegel, Richard
中科院分区：
文献类型：
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作者：
Krawczyk, Ewa;Suprynowicz, Frank A.;Schlegel, Richard
16型人乳头瘤病毒的E5蛋白是一种小的疏水性蛋白，主要定位于内质网（ER）膜。为了确定E5在这些膜中的取向，我们采用了一种不同的洗涤剂渗透技术，该技术利用低浓度洋地黄苷选择性渗透质膜和皂角苷渗透所有细胞膜的能力。然后，我们生成了一个具有生物活性的E5蛋白，在其N和C末端都有表位标记，并确定了在存在和不存在洗涤剂的情况下这些末端对抗体的可及性。在COS细胞和人宫颈外细胞中，E5的C端暴露于细胞质，而N端则局限于内质网的管腔。最后，E5第三跨膜结构域（和末端亲水氨基酸）的缺失导致一个蛋白的C端位于内质网腔内。综上所述，这些拓扑发现与E5是一个3-pass跨膜蛋白的模型以及其C端与细胞质蛋白相互作用的研究相一致。
The E5 protein of human papillomavirus type 16 is a small, hydrophobic protein that localizes predominantly to membranes of the endoplasmic reticulum (ER). To define the orientation of E5 in these membranes, we employed a differential, detergent permeabilization technique that makes use of the ability of low concentrations of digitonin to selectively permeabilize the plasma membrane and saponin to permeabilize all cellular membranes. We then generated a biologically active E5 protein that was epitope tagged at both its N and C termini and determined the accessibility of these termini to antibodies in the presence and absence of detergents. In both COS cells and human ectocervical cells, the C terminus of E5 was exposed to the cytoplasm, whereas the N terminus was restricted to the lumen of the ER. Finally, the deletion of the E5 third transmembrane domain (and terminal hydrophilic amino acids) resulted in a protein with its C terminus in the ER lumen. Taken together, these topology findings are compatible with a model of E5 being a 3-pass transmembrane protein and with studies demonstrating its C terminus interacting with cytoplasmic proteins.