Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion
Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion
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DOI：
10.1128/jb.178.11.3049-3058.1996
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发表时间：
1996-06-01
影响因子：
3.2
通讯作者：
Beckwith, J
中科院分区：
文献类型：
--
作者：
Jander, G;Cronan, JE;Beckwith, J
大肠杆菌生物素连接酶是一种细胞质蛋白，它能特异性地对多种生物体的生物素接受结构域进行生物素化。这种体内生物素化可以作为研究蛋白分泌和膜蛋白插入的敏感信号。当谢尔曼丙酸杆菌转羧化酶（PSBT）的1.3S亚基的生物素接受结构域被翻译融合到质周蛋白碱性磷酸酶和麦糖结合蛋白上时，野生型大肠杆菌PSBT很少或没有生物素化。叠氮化钠对SecA的抑制以及SecB、SecD和SecF的突变均会减慢蛋白质分泌，从而导致PSBT的生物素化。当PSBT与大肠杆菌内膜蛋白MaIF融合时，它作为一种拓扑标记物：与MaIF细胞质域的融合被生物素化，而与质周域的融合通常不会被生物素化。如果叠氮化钠抑制了SecA，或者细胞内的SecE被耗尽，那么MaIF第二质周结构域的插入速度就会减慢，从而导致PSBT在这部分蛋白的融合发生生物素化。与已经用于研究蛋白质易位的其他蛋白质融合相比，PSBT融合的优势在于可以用于研究插入过程的速率。
Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MaIF, it acts as a topological marker: fusions to cytoplasmic domains of MaIF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MaIF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.