Studies on the biosynthesis and secretion of mating pheromone (farnesyl peptide) in a heterobasidiomycetous yeast.

异担子菌酵母球红冬孢酵母的交配型A细胞分泌交配信息素rhodotorucine A，诱导相反交配型细胞的性分化（a）。信息素是一个S-法尼基化的十一肽，具有非常有趣的结构。为了阐明其交配信息素的加工和分泌机制，我们克隆了其交配信息素基因并进行了序列测定。本研究的主要结果如下：1.以根据交配信息素氨基酸序列合成的寡核苷酸为探针，通过筛选A细胞基因组文库，克隆了红曲霉素A基因。从克隆的核苷酸序列中，发现推导的交配信息素的前体蛋白含有4个串联重复的信息素序列，在信息素序列之间有一个间隔序列thr-val-ser-ala。间隔区序列可以用作肽的S-法尼基化的信号。可以 关于我们 在几乎所有分泌蛋白的N-末端发现的用于蛋白分泌的典型信号序列在前体中不存在。从这些结果中，我们提出，rhodotorucine A分泌的一种新的途径，其中修饰的肽的疏水性法呢基残基起着重要的作用。使用免疫印迹分析，在细胞提取物中发现具有预测分子量的多肽。2.在A细胞基因组中发现了3个基因RHA 1、RHA 2和RHA 3，分别编码4、3和5个串联重复的红曲霉素A序列。3. RHA基因仅在交配型A细胞中发现，而在a细胞中未发现。这一结果与已知的酵母信息素基因中的结果大不相同，其中交配信息素基因存在于两种交配类型中。4.根据前体的氨基酸序列合成含有间隔序列的寡肽。使用肽及其类似物测定肽法尼基化的酶促反应。少

Mating type A cells of the heterobasidiomycetous yeast Rhodosporidium toruloides, secrete mating pheromones rhodotorucine A that induce sexual differentiation in the cells of the opposite mating type (a). The pheromone is a S-farnesylated undecapeptide of quite interesting structure. To elucidate the mechanism of processing and secretion of the mating pheromone, we cloned and sequenced the genes for the mating pheromone. Important observations of our study are as follows: l.The genes for rhodotorucine A were cloned by screening genomic library of A cells using oligonucleotides synthesized according to the amino acid sequences of the mating pheromone as probe. From the nucleotide sequences of the clone, it was found that the deduced precursor protein for the mating pheromone contains four tandem repeats of the pheromone sequences, with a spacer sequence thr-val-ser-ala between the pheromone sequences. The spacer sequences may serve as a signal for the S-farnesylation of the peptide. Can … More onical signal sequences for protein secretion found at the N-terminus of almost all secretory proteins were not present in the precursor. From these results, we proposed that rhodotorucine A is secreted by a novel pathway in which modification of the peptide by the hydrophobic farnesyl residue plays an important role. Using immunoblot analysis, a polypeptide with the predicted molecular weight was found in the cell extract. 2.Three genes RHA1, RHA2 and RHA3 that encode four, three and five tandem repeats of rhodotorucine A sequences were found in the genome of A cells. 3.The RHA genes were found only in mating type A cells but not in a cells. The result was quite different from that known in the pheromone genes for Saccharomyces cerevisiar, in which the genes for mating pheromones are present in both mating types. 4.Oligopeptides that contain spacer sequences were synthesized according to the amino acid sequences of the precursor. Using the peptides and its analogues enzymatic reaction for the peetide farnesylation was measured. Less