Chen Minyong, Xie Kun, Nouwen Nico, Driessen Arnold J M, Dalbey Ross E
Department of Chemistry and Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.
J Biol Chem. 2003 Jun 27;278(26):23295-300. doi: 10.1074/jbc.M301008200. Epub 2003 Apr 21.
Conditional lethal YidC mutants have been isolated to decipher the role of YidC in the assembly of Sec-dependent and Sec-independent membrane proteins. We now show that the membrane insertion of the Sec-independent M13 procoat-lep protein is inhibited in a short time in a temperature-sensitive mutant when shifted to the nonpermissive temperature. This provides an additional line of evidence that YidC plays a direct role in the insertion of the Sec-independent M13 procoat protein. However, in the temperature-sensitive mutant, the insertion of the Sec-independent Pf3 phage coat protein and the Sec-dependent leader peptidase were not strongly inhibited at the restricted temperatures. Conversely, using a cold-sensitive YidC strain, we find that the membrane insertion of the Sec-independent Pf3 coat protein is blocked, and the Sec-dependent leader peptidase is inhibited at the nonpermissive temperature, whereas the insertion of the M13 procoat protein is nearly normal. These data show that the YidC function for procoat and its function for Pf3 coat and possibly leader peptidase are genetically separable and suggest that the YidC structural requirements are different for the Sec-independent M13 procoat and Pf3 coat phage proteins that insert by different mechanisms.
为了阐明YidC在依赖Sec和不依赖Sec的膜蛋白组装中的作用，已分离出条件致死性YidC突变体。我们现在表明，在温度敏感型突变体中，当转移到非允许温度时，不依赖Sec的M13原衣壳-lep蛋白的膜插入在短时间内受到抑制。这提供了额外的证据表明YidC在不依赖Sec的M13原衣壳蛋白的插入中起直接作用。然而，在温度敏感型突变体中，不依赖Sec的Pf3噬菌体衣壳蛋白和依赖Sec的前导肽酶的插入在受限温度下并未受到强烈抑制。相反，使用冷敏感型YidC菌株，我们发现不依赖Sec的Pf3衣壳蛋白的膜插入被阻断，并且依赖Sec的前导肽酶在非允许温度下受到抑制，而M13原衣壳蛋白的插入几乎正常。这些数据表明，YidC对原衣壳的功能及其对Pf3衣壳以及可能的前导肽酶的功能在遗传上是可分离的，并表明YidC对通过不同机制插入的不依赖Sec的M13原衣壳和Pf3衣壳噬菌体蛋白的结构要求不同。
