Verstrepen Kevin J, Van Laere Stijn D M, Vercammen Jo, Derdelinckx Guy, Dufour Jean-Pierre, Pretorius Isak S, Winderickx Joris, Thevelein Johan M, Delvaux Freddy R
Centre for Malting and Brewing Science, Department of Food and Microbial Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Leuven (Heverlee), Belgium.
Yeast. 2004 Mar;21(4):367-77. doi: 10.1002/yea.1100.
The yeast alcohol acetyl transferase I, Atf1p, is responsible for the major part of volatile acetate ester production in fermenting Saccharomyces cerevisiae cells. Some of these esters, such as ethyl acetate and isoamyl acetate, are important for the fruity flavours of wine, beer and other fermented beverages. In order to reveal the subcellular localization of Atf1p and further unravel the possible physiological role of this protein, ATF1::GFP fusion constructs were overexpressed in brewer's yeast. The transformant strain showed a significant increase in acetate ester formation, similar to that of an ATF1 overexpression strain, indicating that the Atf1p-GFP fusion protein was active. UV fluorescence microscopy revealed that the fusion protein was localized in small, sphere-like organelles. These organelles could be selectively stained by the fluorescent dye Nile red, indicating that they contained high amounts of neutral lipids and/or sterols, a specific characteristic of yeast lipid particles. Purification of lipid particles from wild type and ATF1 deletion cells confirmed that the Atf1p-GFP fusion protein was located in these organelles. Furthermore, a clear alcohol acetyl transferase activity could be measured in the purified lipid particles of both wild type and transformed cells. The localization of Atf1p in lipid particles may indicate that Atf1p has a specific role in the lipid and/or sterol metabolism that takes place in these particles.
酵母乙醇乙酰转移酶I（Atf1p）负责酿酒酵母发酵细胞中大部分挥发性乙酸酯的产生。其中一些酯类，如乙酸乙酯和乙酸异戊酯，对葡萄酒、啤酒和其他发酵饮料的果香很重要。为了揭示Atf1p的亚细胞定位，并进一步阐明该蛋白可能的生理作用，在酿酒酵母中过表达了ATF1::GFP融合构建体。转化菌株的乙酸酯形成显著增加，类似于ATF1过表达菌株，表明Atf1p-GFP融合蛋白具有活性。紫外荧光显微镜显示，融合蛋白定位于小的球状细胞器中。这些细胞器可以被荧光染料尼罗红选择性染色，表明它们含有大量中性脂质和/或固醇，这是酵母脂质颗粒的一个特定特征。从野生型和ATF1缺失细胞中纯化脂质颗粒证实，Atf1p-GFP融合蛋白位于这些细胞器中。此外，在野生型和转化细胞的纯化脂质颗粒中都可以检测到明显的乙醇乙酰转移酶活性。Atf1p在脂质颗粒中的定位可能表明Atf1p在这些颗粒中发生的脂质和/或固醇代谢中具有特定作用。
