Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe
Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe
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裂殖酵母中的液泡蛋白分选：裂殖酵母中羧肽酶 Y 的克隆、生物合成、运输和加工
DOI：
10.1128/jb.179.13.4179-4189.1997
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发表时间：
1997
影响因子：
3.2
通讯作者：
K. Takegawa
中科院分区：
文献类型：
--
作者：
M. Tabuchi;Osamu Iwaihara;Yoshihiko Ohtani;Nobuhiro Ohuchi;Jun Ichiro Sakurai;Toshie Morita;S. Iwahara;K. Takegawa
PCR 用于从裂殖酵母裂殖酵母中分离羧肽酶 Y (CPY) 同源基因。克隆的粟酒裂殖酵母 cpy1+ 基因具有单个开放阅读框，编码 950 个氨基酸，具有一个潜在的 N-糖基化位点。它似乎是作为一种无活性的前原蛋白合成的，可能在易位到适当的细胞内细胞器后进行加工。 C 末端成熟区在其他丝氨酸羧肽酶中高度保守。相比之下，来自酿酒酵母的 CPY 中含有液泡分选信号的 N 端原区显示出较少的相同残基。原区域包含两个不寻常的重复序列；重复序列I由每个13个氨基酸的7个连续重复片段组成，并且重复序列II由每个9个氨基酸的7个连续重复片段组成。脉冲追踪放射性标记分析表明，Cpy1p 最初以 110 kDa 前体形式合成，并通过去除其前体片段的 51 kDa 单多肽链中间体形式最终转化为异二聚体（成熟形式），在还原条件下通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测为 32 kDa 蛋白质。与酿酒酵母 CPY 一样，粟酒裂殖酵母 Cpy1p 不需要 N 连接寡糖部分来进行液泡递送。为了研究粟酒裂殖酵母 Cpy1p 的液泡分选信号，我们构建了 cpy1+-SUC2 基因融合体，该融合蛋白指导由不同长度的粟酒裂殖酵母 Cpy1p 的 N 端片段与分泌酶酿酒酵母转化酶融合组成的杂合蛋白的合成。 Cpy1 的 N 端 478 个氨基酸足以引导 Cpy1-Inv 杂合蛋白递送至液泡。这些结果表明Cpy1的前肽含有假定的液泡分选信号。
PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.
DOI：
--
发表时间：
1985
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Dieckmann,CL;Tzagoloff,A
通讯作者：
Tzagoloff,A
DOI：
10.1002/j.1460-2075.1989.tb03614.x
发表时间：
1989-07
期刊：
The EMBO Journal
影响因子：
--
作者：
J. Rothman;I. Howald;T. Stevens
通讯作者：
J. Rothman;I. Howald;T. Stevens
酵母羧肽酶 Y 需要糖基化才能实现有效的细胞内转运，但不需要糖基化来实现液泡分选、体内稳定性或活性。
DOI：
10.1111/j.1432-1033.1991.tb15959.x
发表时间：
1991
期刊：
European journal of biochemistry
影响因子：
--
作者：
Winther,JR;Stevens,TH;Kielland-Brandt,MC
通讯作者：
Kielland-Brandt,MC