The effects of palmitoylation on membrane association of Semliki Forest virus RNA capping enzyme
The effects of palmitoylation on membrane association of Semliki Forest virus RNA capping enzyme
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DOI：
10.1074/jbc.271.45.28567
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发表时间：
1996-11-08
影响因子：
4.8
通讯作者：
Kaariainen, L
中科院分区：
文献类型：
--
作者：
Laakkonen, P;Ahola, T;Kaariainen, L
塞姆利基森林病毒的非结构蛋白Nsp 1具有鸟嘌呤-7-甲基转移酶和鸟苷酰转移酶样活性，这是病毒mRNA加帽所需的。它是棕榈酰化的，与质膜、内体和溶酶体的细胞质表面紧密结合。为了定位酰化位点和推定的膜靶向结构域，在nsp 1基因中进行了大量缺失。大多数缺失导致非棕榈酰化，酶活性，细胞质蛋白的表达。棕榈酸可以从Nsp 1释放与中性羟胺，表明硫酯连接到半胱氨酸残基，因此，我们突变Nsp 1的保守半胱氨酸残基为丙氨酸。Cys（418）、Cys（419）和Cys（420）的三重突变导致非棕榈酰化的Nsp 1，其具有酶活性并且仍然与膜结合。然而，它可以从1 M NaCl的膜释放，而50 mm碳酸钠（pH 12）需要释放野生型Nsp 1，这表明从一个完整的外周膜蛋白的转换。间接共聚焦免疫荧光显微镜显示，nonpalmitoylated Nsp 1与质膜标志物，刀豆球蛋白A共定位。然而，在丝状伪足中没有检测到，丝状伪足在表达野生型Nsp 1的细胞中被严重染色。这些结果表明，Nsp 1的酰化不是其靶向质膜所必需的，但它是迁移到质膜的丝状伪足延伸所必需的。
The nonstructural protein Nsp1 of Semliki Forest virus has guanine-7-methyltransferase and guanylyltransferase-like activities, required in the capping of viral mRNAs. It is palmitoylated and tightly associated with the cytoplasmic surface of the plasma membrane, endosomes, and lysosomes, To localize the acylation site(s) and the putative membrane-targeting domain, a number of deletions were made in the nsp1 gene. Most deletions resulted in the expression of nonpalmitoylated, enzymatically inactive, cytoplasmic protein. Palmitate could be released from Nsp1 with neutral hydroxylamine, indicating a thioester linkage to a cysteine residue, Therefore we mutated the conserved cysteine residues of Nsp1 to alanine. Triple mutation of Cys(418), Cys(419), and Cys(420) resulted in nonpalmitoylated Nsp1, which was enzymatically active and still associated with membranes. However, it could be released from the membranes with 1 M NaCl, whereas 50 mm sodium carbonate (pH 12) was required to release wild type Nsp1, suggesting a conversion from an integral to a peripheral membrane protein, Indirect confocal immunofluorescence microscopy showed that the nonpalmitoylated Nsp1 colocalized with the plasma membrane marker, concanavalin A. However, it was not detected in filopodia, which were heavily stained in cells expressing wild type Nsp1. These results indicate that the acylation of Nsp1 was not needed for its targeting to the plasma membrane, but it was necessary for the migration to the filopodial extensions of the plasma membrane.