Gallusser A, Kuhn A
Microbiology Department, University of Basel, Switzerland.
EMBO J. 1990 Sep;9(9):2723-9. doi: 10.1002/j.1460-2075.1990.tb07459.x.
Bacteriophage M13 procoat protein is synthesized on free polysomes prior to its assembly into the inner membrane of Escherichia coli. As an initial step of the membrane insertion pathway, the precursor protein interacts with the cytoplasmic face of the inner membrane. We have used oligonucleotide-directed mutagenesis to study the regions of the procoat protein involved in membrane binding. We find that there is an absolute requirement for positively charged amino acids at both ends of the protein. Replacing these with negatively charged residues resulted in an accumulation of the precursor in the cytoplasm. We propose that the positively charged amino acids are directly involved in membrane binding, possibly directly to the negatively charged phospholipid head groups. This was tested in vitro with artificial liposomes. Whereas wild-type procoat interacted with these liposomes, we found that procoat mutants with negatively charged amino acids at both ends did not bind. Therefore, we conclude that newly synthesized M13 procoat protein binds electrostatically to the negatively charged inner membrane of E. coli.
噬菌体M13原衣壳蛋白在组装到大肠杆菌内膜之前在游离多聚核糖体上合成。作为膜插入途径的第一步，前体蛋白与内膜的细胞质面相互作用。我们使用寡核苷酸定向诱变来研究原衣壳蛋白中参与膜结合的区域。我们发现蛋白质两端对带正电荷的氨基酸有绝对需求。用带负电荷的残基取代这些氨基酸会导致前体在细胞质中积累。我们提出带正电荷的氨基酸直接参与膜结合，可能直接与带负电荷的磷脂头部基团结合。这在体外用人造脂质体进行了测试。野生型原衣壳与这些脂质体相互作用，而我们发现两端带负电荷氨基酸的原衣壳突变体不结合。因此，我们得出结论，新合成的M13原衣壳蛋白通过静电作用与大肠杆菌带负电荷的内膜结合。
