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首页> 外文期刊>Molecular and Cellular Biology >Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product.
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Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product.

机译：酵母分泌机制的一个组成部分的特征：SEC18基因产物的鉴定。

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SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind transiently to small vesicles such as those presumed to participate in secretory protein transport between ER and the Golgi complex.
机译：内质网（ER）与高尔基体之间的分泌蛋白转运需要SEC18基因功能。我们通过补充sec18-1突变克隆了SEC18基因。基因破坏表明，SEC18对酵母细胞的生长至关重要。基因的序列分析揭示了一个2,271个碱基对的开放阅读框，其可编码83.9千道尔顿的蛋白质。预测的蛋白质序列与其他已知蛋白质序列没有显着相似性。 SEC18的体外转录和翻译导致合成了大约84和82道尔顿的两种蛋白质。针对Sec18-β-半乳糖苷酶融合蛋白产生的抗血清还检测到35S标记酵母细胞提取物中的两种蛋白质（统称为Sec18p），其大小与体外翻译所见相同。 SEC18 mRNA 5'末端的定位图仅显示了一个主要的转录起始位点，这表明Sec18p的多种形式并非来自具有不同5'末端的mRNA。脉冲追踪实验的结果表明，两种形式的Sec18p并非翻译后加工的结果。我们建议在不同的框架内AUG起始密码子处启动翻译可能是两种形式的Sec18p的存在。疏水性分析表明，蛋白质本质上是亲水性的，并且没有任何可以预测用作信号序列或跨膜锚的区域。尽管在Sec18p序列中存在N-联糖基化的潜在位点，但体内SEC18基因产物的大小不受衣霉素的影响，表明Sec18p没有进入分泌途径。这些结果表明Sec18p驻留在细胞质中。初步的细胞分离研究表明Sec18p与ER或高尔基体不相关，但观察到与100,000 x g沉淀级分相关。这表明Sec18p可能与小囊泡暂时结合，例如假定参与ER和高尔基体之间分泌蛋白转运的小囊泡。

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