Pardo M, Ward M, Bains S, Molina M, Blackstock W, Gil C, Nombela C
Departmento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Spain.
Electrophoresis. 2000 Oct;21(16):3396-410. doi: 10.1002/1522-2683(20001001)21:16<3396::AID-ELPS3396>3.0.CO;2-J.
In fungi, cell shape is determined by the presence of a rigid cell wall which separates the cell from the extracellular medium. This highly dynamic structure is essential for the maintenance of cell integrity and is involved in several phenomena such as flocculation, adherence and pathogenicity. The composition of the fungal cell wall is well known, but issues such as the assembly and remodeling of its components remain poorly understood. In an attempt to study the de novo construction of the yeast cell wall, we have undertaken a large-scale proteomic approach to analyze the proteins secreted by regenerating protoplasts. Upon incubation of protoplasts in regenerating conditions, numerous proteins are secreted into the culture medium. These presumably include proteins destined for the cell wall, comprising both structural proteins as well as enzymes involved in cell wall biogenesis. This work reports the establishment of a reference map of proteins secreted by regenerating protoplasts by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and their identification by mass spectrometry. Thirty-two different proteins have been identified, including known cell wall proteins, glycolytic enzymes, heat shock proteins, and proteins involved in several other processes. Using this approach, novel proteins possibly involved in cell wall construction have also been identified. This reference map will allow comparative analyses to be carried out on a selected collection of mutants affected in the cell wall.
在真菌中，细胞形状由一层坚硬的细胞壁决定，该细胞壁将细胞与细胞外介质分隔开来。这种高度动态的结构对于维持细胞完整性至关重要，并参与了诸如絮凝、黏附及致病性等多种现象。真菌细胞壁的组成是众所周知的，但其成分的组装和重塑等问题仍知之甚少。为了研究酵母细胞壁的从头构建，我们采用了大规模蛋白质组学方法来分析再生原生质体分泌的蛋白质。将原生质体在再生条件下孵育时，大量蛋白质会分泌到培养基中。这些蛋白质可能包括 destined for the cell wall，既有结构蛋白，也有参与细胞壁生物合成的酶。这项工作报告了通过二维聚丙烯酰胺凝胶电泳（2-D PAGE）建立再生原生质体分泌蛋白质的参考图谱，并通过质谱对其进行鉴定。已鉴定出 32 种不同的蛋白质，包括已知的细胞壁蛋白、糖酵解酶、热休克蛋白以及参与其他几个过程的蛋白质。利用这种方法，还鉴定出了可能参与细胞壁构建的新蛋白质。该参考图谱将允许对一组选定的细胞壁相关突变体进行比较分析。
