Veit M, Reverey H, Schmidt M F
Institut für Immunologie und Molekularbiologie (IMB), Freie Universität Berlin, Germany.
Biochem J. 1996 Aug 15;318 ( Pt 1)(Pt 1):163-72. doi: 10.1042/bj3180163.
We report remarkable differences in the fatty acid content of thioester-type acylated glycoproteins of enveloped viruses from mammalian cells. The E2 glycoprotein of Semliki Forest virus contains mainly palmitic acid like most other palmitoylated proteins analysed so far. However, the other glycoprotein (E1) of the same virus, as well as the HEF (haemagglutinin esterase fusion) glycoprotein of influenza C virus, are unique in this respect because they are acylated primarily with stearic acid. Comparative radiolabelling of uninfected cells with different fatty acids suggests that stearate may also be the prevailing fatty acid in some cellular acylproteins. To look for further differences between palmitoylated and stearoylated glycoproteins we characterized stearoylation in more detail. We identified the acylation site of HEF as a cysteine residue located at the boundary between the transmembrane region and the cytoplasmic tail. The attachment of stearate to HEF and E1 occurs post-translationally in a pre-Golgi compartment. Thus, stearoylated and palmitoylated proteins cannot be discriminated on the basis of the fatty acid linkage site or the intracellular compartment, where acylation occurs. However, stearoylated acylproteins contain a very short, positively charged cytoplasmic tail, whereas in palmitoylated proteins this molecular region is longer. Replacing the short cytoplasmic tail of stearoylated HEF with the long influenza A virus haemagglutinin (HA) tail in an HEF-HA chimera, and subsequent vaccinia T7 expression in CV-1 cells, yielded proteins with largely palmitic acid bound. The reverse chimera, HA-HEF with a short cytoplasmic tail was not fatty acylated at all during expression, indicating that conformational or topological constraints control fatty acid transfer.
我们报道了来自哺乳动物细胞的包膜病毒硫酯型酰化糖蛋白脂肪酸含量的显著差异。辛德毕斯病毒的E2糖蛋白主要含有棕榈酸，这与迄今为止分析的大多数其他棕榈酰化蛋白类似。然而，同一病毒的另一种糖蛋白（E1）以及丙型流感病毒的血凝素酯酶融合（HEF）糖蛋白在这方面是独特的，因为它们主要被硬脂酸酰化。用不同脂肪酸对未感染细胞进行比较放射性标记表明，硬脂酸也可能是某些细胞酰基蛋白中的主要脂肪酸。为了寻找棕榈酰化和硬脂酰化糖蛋白之间的进一步差异，我们更详细地对硬脂酰化进行了表征。我们确定HEF的酰化位点是位于跨膜区和细胞质尾部边界的一个半胱氨酸残基。硬脂酸与HEF和E1的连接发生在翻译后，在高尔基体前区室中。因此，不能根据脂肪酸连接位点或酰化发生的细胞内区室来区分硬脂酰化和棕榈酰化蛋白。然而，硬脂酰化酰基蛋白含有非常短的带正电荷的细胞质尾部，而在棕榈酰化蛋白中，这个分子区域更长。在HEF-HA嵌合体中，用甲型流感病毒血凝素（HA）的长尾巴取代硬脂酰化HEF的短细胞质尾部，随后在CV-1细胞中进行痘苗T7表达，产生了大量结合棕榈酸的蛋白质。反向嵌合体，即具有短细胞质尾部 的HA-HEF在表达过程中根本没有发生脂肪酸酰化，这表明构象或拓扑限制控制着脂肪酸转移。
