Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
Microb Cell Fact. 2011 May 19;10:37. doi: 10.1186/1475-2859-10-37.
The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach.
Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response.
Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway.
人类病毒表面蛋白以及其他哺乳动物糖蛋白在高等真核细胞中的表达效率远高于酵母。然而，人们对于病毒表面糖蛋白在酵母中高效表达的限制因素还知之甚少。为了确定可能的瓶颈，我们对重组腮腺炎血凝素神经氨酸酶（MuHN）和麻疹血凝素（MeH）在酿酒酵母中的过表达进行了详细研究，将重组蛋白分析与蛋白质组学方法相结合。
过表达的重组 MuHN 和 MeH 蛋白以大聚集体的形式存在，在天然条件下无活性且完全不溶。此外，大部分重组蛋白以未糖基化的不成熟前体形式存在。酵母裂解物的分级分离表明，病毒表面蛋白聚集体的核心由 MuHN 或 MeH 二硫键连接的多聚体组成，涉及真核翻译延伸因子 1A（eEF1A），并与小热休克蛋白（sHsps）紧密相关，只有在变性条件下才能将其去除。大 Hsp 复合物似乎作为聚集体核心的外围结合物存在，因为它们可以在高盐浓度下轻易去除。蛋白质组学分析表明，未糖基化的病毒蛋白前体的积累导致酵母细胞中特定的细胞质未折叠蛋白反应（UPR-Cyto），其特征为小 Hsps 与 Hsp70、Hsp90 和 Hsp110 家族的大伴侣蛋白的不同作用和调节。与大多数环境压力不同，在合成重组 MuHN 和 MeH 的反应中，只有大 Hsp 被上调，而 sHsp 则没有。有趣的是，eEF1A 的数量在这种应激反应中也增加了。
MuHN 和 MeH 前体穿过内质网膜的低效易位是酵母中高水平表达的瓶颈。这些重组蛋白的过表达诱导了细胞质未折叠蛋白反应的细胞对应物，即 UPR-Cyto，它代表了参与热休克反应的一组蛋白。eEF1A 的参与可能解释了为什么只有大伴侣蛋白而不是 sHsp 在这种应激反应中被上调。我们的研究强调了病毒表面蛋白在酵母和哺乳动物细胞分泌途径的第一阶段表达的重要差异。
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