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

异种酵母Rhodosporidium toruloides的交配型A细胞分泌交配信息素rhodotorucine A，诱导异性交配型细胞的性分化(A)。信息素是一种结构非常有趣的s -法酰化的非肽。为了阐明交配信息素的加工和分泌机制，我们克隆并测序了交配信息素的基因。本研究的主要观察结果如下：1 .利用根据交配信息素氨基酸序列合成的寡核苷酸为探针，筛选A细胞基因组文库，克隆了红托鲁氨酸A基因。从该克隆的核苷酸序列中发现，推断出的交配信息素前体蛋白包含4个信息素序列的串联重复序列，信息素序列之间有一个间隔序列。间隔序列可作为肽s -法酰化的信号。在几乎所有的分泌蛋白的n端发现了更多的蛋白质分泌的电子信号序列，而在前体中不存在。根据这些结果，我们提出rhodotorucine A是通过一种新的途径分泌的，其中疏水性法尼基残基对肽的修饰起着重要作用。利用免疫印迹分析，在细胞提取物中发现了具有预测分子量的多肽。2.在A细胞基因组中发现了编码rhodotorucine A序列4、3和5个串联重复序列的基因RHA1、RHA2和RHA3。3.RHA基因只在交配型A细胞中发现，而在A细胞中没有发现。结果与已知的酿酒酵母菌的信息素基因有很大的不同，在酿酒酵母菌中，交配信息素的基因在两种交配类型中都存在。4.根据前体的氨基酸序列合成含有间隔序列的寡肽。利用该多肽及其类似物对酶促反应进行了法尼化测定。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