Schenk B, Rush J S, Waechter C J, Aebi M
Institute for Microbiology, ETH Zurich, CH-8092 Zurich, Switzerland.
Glycobiology. 2001 Jan;11(1):89-98. doi: 10.1093/glycob/11.1.89.
Dolichyl monophosphate (Dol-P) is a polyisoprenoid glycosyl carrier lipid essential for the assembly of a variety of glycoconjugates in the endoplasmic reticulum of eukaryotic cells. In yeast, dolichols with chain lengths of 14--17 isoprene units are predominant, whereas in mammalian cells they contain 19--22 isoprene units. In this biosynthetic pathway, t,t-farnesyl pyrophosphate is elongated to the appropriate long chain polyprenyl pyrophosphate by the sequential addition of cis-isoprene units donated by isopentenyl pyrophosphate with t,t,c-geranylgeranyl pyrophosphate being the initial intermediate formed. The condensation steps are catalyzed by cis-isoprenyltransferase (cis-IPTase). Genes encoding cis-IPTase activity have been identified in Micrococcus luteus, Escherichia coli, Arabidopsis thaliana, and Saccharomyces cerevisiae (RER2). Yeast cells deleted for the RER2 locus display a severe growth defect, but are still viable, possibly due to the activity of an homologous locus, SRT1. The dolichol and Dol-P content of exponentially growing revertants of RER2 deleted cells (Delta rer2) and of cells overexpressing SRT1 have been determined by HPLC analysis. Dolichols and Dol-Ps with 19--22 isoprene units, unusually long for yeast, were found, and shown to be utilized for the biosynthesis of lipid intermediates involved in protein N-glycosylation. In addition, cis-IPTase activity in microsomes from Delta rer2 cells overexpressing SRT1 was 7- to 17-fold higher than in microsomes from Delta rer2 cells. These results establish that yeast contains at least two cis-IPTases, and indicate that the chain length of dolichols is determined primarily by the enzyme catalyzing the chain elongation stage of the biosynthetic process.
多萜醇单磷酸酯（Dol-P）是一种聚异戊二烯糖基载体脂质，对于真核细胞内质网中多种糖缀合物的组装至关重要。在酵母中，链长为14 - 17个异戊二烯单元的多萜醇占主导，而在哺乳动物细胞中它们含有19 - 22个异戊二烯单元。在这条生物合成途径中，反，反-法呢基焦磷酸通过依次添加由异戊烯基焦磷酸提供的顺式异戊二烯单元而延伸为适当的长链聚异戊烯焦磷酸，反，反，顺-香叶基香叶基焦磷酸是形成的初始中间体。缩合步骤由顺式异戊烯基转移酶（cis-IPTase）催化。已在藤黄微球菌、大肠杆菌、拟南芥和酿酒酵母（RER2）中鉴定出编码cis-IPTase活性的基因。缺失RER2基因座的酵母细胞表现出严重的生长缺陷，但仍可存活，这可能是由于同源基因座SRT1的活性。已通过高效液相色谱分析确定了RER2缺失细胞（Δrer2）的指数生长回复株和过表达SRT1的细胞中的多萜醇和Dol-P含量。发现了具有19 - 22个异戊二烯单元的多萜醇和Dol-P，这对酵母来说异常长，并表明它们被用于参与蛋白质N-糖基化的脂质中间体的生物合成。此外，过表达SRT1的Δrer2细胞微粒体中的cis-IPTase活性比Δrer2细胞微粒体中的高7至17倍。这些结果表明酵母至少含有两种cis-IPTase，并表明多萜醇的链长主要由催化生物合成过程中链延伸阶段的酶决定。
