Shimoike T, Taura T, Kihara A, Yoshihisa T, Akiyama Y, Cannon K, Ito K
Department of Cell Biology, Kyoto University, Japan.
J Biol Chem. 1995 Mar 10;270(10):5519-26. doi: 10.1074/jbc.270.10.5519.
A mutant form of SecY, SecY-d1, was previously suggested to sequester a component(s) of the protein translocator complex. Its synthesis from a plasmid leads to interference with protein export in Escherichia coli. SecE is a target of this sequestration, and its overproduction cancels the export interference. We now report that overexpression of another gene, termed syd, also suppresses secY-d1. The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino-terminal sequence. Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane. SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY. SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available. Overproduction of Syd was found to stabilize oversynthesized SecY. However, Syd cannot stabilize the SecY-d1 form of SecY. Thus, in the presence of both secY+ and secY-d1, Syd increases the effective SecY+/SecY-d1 ratio in the cell and cancels the dominant interference by the latter. We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened. These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY. Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd.
之前有人提出，SecY的一种突变形式SecY-d1会隔离蛋白质转运体复合物的一个或多个组分。从质粒合成该蛋白会干扰大肠杆菌中的蛋白质输出。SecE是这种隔离作用的一个靶点，其过量表达可消除输出干扰。我们现在报告，另一个名为syd的基因的过表达也能抑制secY-d1。syd的核苷酸序列预测它编码一个含有181个氨基酸残基的蛋白质，该蛋白已通过过量表达、纯化及氨基末端序列测定得以鉴定。细胞分级分离实验表明，Syd与细胞质膜的细胞质表面松散结合。SecY可能参与了Syd与膜的结合，因为这种结合是可饱和的，其程度取决于SecY的过量表达。SecY在体内会迅速降解，除非其主要伴侣SecE充足可用。发现Syd的过量表达能稳定过量合成的SecY。然而，Syd不能稳定SecY的SecY-d1形式。因此，在secY+和secY-d1同时存在的情况下，Syd会增加细胞中有效SecY+/SecY-d1的比例，并消除后者的显性干扰。我们还发现，Syd的过量表达会显著抑制secY24突变体细胞中的蛋白质输出，在该突变体细胞中SecY-SecE相互作用已被削弱。这些结果表明，Syd尤其是在过量表达时，具有与SecY相互作用的能力。结合syd基因破坏后明显缺乏表型后果，讨论了这种相互作用可能的意义。
