Boyd D, Beckwith J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1989 Dec;86(23):9446-50. doi: 10.1073/pnas.86.23.9446.
When alkaline phosphatase is fused to the periplasmic domain of a cytoplasmic membrane protein, it is efficiently exported to the periplasm. Such a hybrid protein exhibits high alkaline phosphatase enzymatic activity. When alkaline phosphatase is fused to the cytoplasmic domain of a membrane protein, it remains, for the most part, in the cytoplasm. Such fusions exhibit low enzymatic activity. However, stable retention of alkaline phosphatase in the cytoplasm requires the presence in the fusion protein of the cytoplasmic loop ordinarily present in that position in the native, unfused protein. Using oligonucleotide-directed mutagenesis, we have shown that positively charged amino acids are required for the stable cytoplasmic localization of the fused alkaline phosphatase. We propose that, in addition to hydrophobic transmembrane segments, positively charged amino acids in the hydrophilic cytoplasmic domains of a membrane protein are determinants of the protein's topology.
当碱性磷酸酶与细胞质膜蛋白的周质结构域融合时，它能有效地输出到周质中。这样的杂合蛋白表现出高碱性磷酸酶酶活性。当碱性磷酸酶与膜蛋白的细胞质结构域融合时，它在很大程度上仍留在细胞质中。这种融合表现出低酶活性。然而，碱性磷酸酶在细胞质中的稳定保留需要融合蛋白中存在天然未融合蛋白该位置通常存在的细胞质环。利用寡核苷酸定向诱变，我们已经表明带正电荷的氨基酸是融合碱性磷酸酶稳定细胞质定位所必需的。我们提出，除了疏水跨膜区段外，膜蛋白亲水性细胞质结构域中的带正电荷氨基酸是蛋白质拓扑结构的决定因素。
