Hattori T, Ichihara S, Nakamura K
Eur J Biochem. 1987 Aug 3;166(3):533-8. doi: 10.1111/j.1432-1033.1987.tb13546.x.
A precursor for sporamin A, the storage protein of the tuberous roots of sweet potato deposited in the vacuole, is synthesized on membrane-bound polysomes and has an extra peptide of 37 amino acids at the N-terminus of the mature form, which can be divided into an N-terminal putative signal peptide sequence (residues -37 to -17) and a segment enriched with charged amino acids (residues -16 to -1) [Hattori, T., et al. (1985) Plant Mol. Biol. 5, 313-320]. We examined the in vitro processing of the sporamin A precursor using a messenger RNA derived from a full-length cDNA by the SP6 transcription system. When the in vitro translation in a wheat germ cell-free system was carried out in the presence of dog pancreas microsomal membranes, the precursor polypeptide (Mr = 24,000) was processed into an intermediate form still larger than the mature polypeptide (Mr = 20,000). The processed intermediate form was also produced by addition of microsomal membranes from sweet potato and potato in the translation reaction, although less efficiently compared to dog membranes. Moreover, Escherichia coli cells expressing sporamin precursor accumulated a polypeptide with the same electrophoretic mobility as the intermediate form produced in vitro. The processing by dog membranes is accompanied by translocation of the polypeptide across the membranes as assayed by resistance to externally added proteases. The N-terminal amino acid sequencing analysis of [3H]leucine-labelled intermediate form produced in vitro by dog membranes indicated that co-translational processing of the sporamin precursor by endoplasmic reticulum membranes removes only the signal peptide segment from the extra peptide, and suggested that the charged segment following the signal peptide is removed post-translationally during the transport of sporamin into vacuole. The significance of two-step processing of plant vacuolar protein precursor is discussed in relation to the two-step processing of precursors for yeast vacuolar proteins and animal lysosomal proteins.
红薯块根中储存于液泡的贮藏蛋白sporamin A的前体，是在膜结合多核糖体上合成的，在成熟形式的N端有一段37个氨基酸的额外肽段，该肽段可分为N端假定信号肽序列（-37至-17位残基）和富含带电荷氨基酸的片段（-16至-1位残基）[服部彻等人，（1985年）《植物分子生物学》5，313 - 320]。我们使用通过SP6转录系统从全长cDNA获得的信使RNA，研究了sporamin A前体的体外加工过程。当在小麦胚无细胞系统中进行体外翻译时，若存在犬胰腺微粒体膜，前体多肽（Mr = 24,000）会被加工成一种仍比成熟多肽（Mr = 20,000）大的中间形式。在翻译反应中加入红薯和马铃薯的微粒体膜，也能产生加工后的中间形式，不过与犬膜相比效率较低。此外，表达sporamin前体的大肠杆菌细胞积累了一种与体外产生的中间形式具有相同电泳迁移率的多肽。犬膜的加工过程伴随着多肽跨膜转运，这可通过对外源添加蛋白酶的抗性来检测。对犬膜体外产生的[³H]亮氨酸标记中间形式进行N端氨基酸测序分析表明，内质网对sporamin前体的共翻译加工仅从额外肽段中去除信号肽片段，这表明信号肽之后的带电荷片段在sporamin转运至液泡的过程中是在翻译后被去除的。本文结合酵母液泡蛋白前体和动物溶酶体蛋白前体的两步加工过程，讨论了植物液泡蛋白前体两步加工的意义。
