Spriggs M K, Collins P L
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Cell Biol. 1990 Jul;111(1):31-44. doi: 10.1083/jcb.111.1.31.
Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.
通过重组SV - 40病毒在组织培养中表达了3型副流感病毒氨基末端锚定（II型取向）血凝素神经氨酸酶（HN）蛋白的六个氨基末端缺失突变体。这些突变包括细胞质结构域的逐步缺失，在某些情况下还包括疏水信号/锚定序列的缺失。信号/锚定序列的三种活性被分离出来：膜插入、转运和锚定/运输。缺乏整个细胞质尾巴的HN蛋白能有效地插入内质网的膜中，但转运到内质网腔的效率较低。然而，成功转运的少量蛋白随后的加工方式与亲本HN蛋白并无区别。因此，这种II型糖蛋白的成熟不需要细胞质结构域。向膜锚定序列的逐步缺失恢复了有效的转运，表明氨基末端的44个氨基酸对于膜插入和转运完全是可有可无的，并且一个10个氨基酸的疏水信号序列对于这两种活性就足够了。通过血凝活性、与构象特异性抗血清的反应性、分子内二硫键的正确形成和同源寡聚化分析，这些后来的HN分子似乎正确折叠。然而，这些分子中的大多数（85 - 90%）积聚在内质网中。这表明折叠和寡聚形成生物活性形式（大概代表病毒粒子刺突）基本上在该隔室内完成，但不足以通过胞吐途径进行有效运输。蛋白质运输似乎也取决于膜锚定序列的结构。这些后来的突变体没有稳定地整合到膜中，通过胞吐途径加工的小部分（10 - 15%）被分泌出来。这里描述的II型糖蛋白的成熟步骤和一些突变效应类似于先前对原型I型糖蛋白的观察结果，表明这两种膜取向的蛋白质在这些过程中有密切的相似性。
