López-Villar Elena, Monteoliva Lucía, Larsen Martin R, Sachon Emmanuelle, Shabaz Mohammed, Pardo Mercedes, Pla Jesús, Gil Concha, Roepstorff Peter, Nombela César
Departamento de Microbiología II, Facultad de Farmacia, UCM, Madrid, Spain.
Proteomics. 2006 Apr;6 Suppl 1:S107-18. doi: 10.1002/pmic.200500479.
Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol that includes sodium carbonate to prevent cytoplasmic contamination. These proteins were analyzed by SDS-PAGE, trypsin digestion and LC-MS/MS for peptide identification. Elongation factors, mitochondrial membrane proteins and a mannosyltransferase involved in cell wall mannan biosynthesis were also identified in this fraction.
尽管烯醇化酶、其他糖酵解酶以及多种缺乏N端分泌信号的细胞质蛋白在酵母和哺乳动物细胞中被广泛描述为位于细胞表面，但其在该外部位置的存在仍存在争议。在此，我们报告不同的实验方法（遗传学、细胞生物学和蛋白质组学）表明酵母烯醇化酶能够到达细胞表面，并描述了参与其细胞表面靶向的蛋白质区域。将杂交烯醇化酶截短体在其C端与酵母内部转化酶或绿色荧光蛋白（GFP）融合作为报告蛋白，结果证明169个N端氨基酸足以将该蛋白靶向到细胞表面。此外，烯醇化酶-GFP融合蛋白与一种质膜标记物共定位。在通过包括碳酸钠以防止细胞质污染的纯化方案获得的膜蛋白中也鉴定出了烯醇化酶。通过SDS-PAGE、胰蛋白酶消化和LC-MS/MS对这些蛋白进行分析以鉴定肽段。在该组分中还鉴定出了延伸因子、线粒体膜蛋白以及参与细胞壁甘露聚糖生物合成的一种甘露糖基转移酶。
