Leu W M, Chen L Y, Liaw L L, Lee Y H
Institute of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China.
J Biol Chem. 1992 Oct 5;267(28):20108-13.
The tyrosinase of Streptomyces antibioticus is encoded by the second open reading frame, melC2 of the melanin operon (melC). The upstream open reading frame melC1 specifies a 146-amino acid protein with a typical NH2-terminal signal-peptide characteristic of a secretory protein. The MelC1 protein is involved in the transfer of copper ion to apotyrosinase MelC2 via binary complex formation (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81; Chen, L.-Y., Leu, W.-M., Wang, K.-T., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 20100-20107). To investigate whether the export of tyrosinase is also dependent on MelC1, a mutational study of its signal-peptide sequence was performed. Four different mutants were obtained. Mutation at the positively charged region (mutant M-6LE, Arg6-Arg7----Leu6-Glu7) or the hydrophobic region (mutant M-16D, Val16----Asp16) led to Mel- phenotypes. These lesions caused a severe 7-10-fold reduction of the export of both the MelC1 and MelC2 proteins and a concomitant accumulation of the two proteins in the cytosolic fraction. The cell-associated tyrosinase activity in M-6LE but not in the M-16D mutant was dramatically reduced to 4% of the activity found in the wild type strain, suggesting that the basic NH2 terminus of MelC1 is also important for the trans-activation function of this protein. Nevertheless, the defects on the trans-activation and/or secretory functions of MelC1 in mutants M-6LE and M-16D are not due to the impairment of the formation of the MelC1.MelC2 complex. The translation of melanin operon genes in these two mutants also decreased. In contrast, the tyrosinase activity and the secretion of MelC2 were not affected if the mutations occurred at the putative cleavage site of the signal peptidase (e.g. mutant M-29SM, Arg29-Ala30----Ser29-Met30 or mutant 29-SMG, Arg29-Ala30-Asp31----Ser29-Med30-Gly31+ ++). Additionally, tyrosinase activity and its export were abolished in a MelC1-negative mutant, M-950. Taken together, these results demonstrate that a functional MelC1 is essential for tyrosinase secretion and activity. Furthermore, the results suggest that like other secretory proteins, basic and hydrophobic residues in the MelC1 signal sequence are an important feature of the signal-peptide and play a pivotal role in the secretion of both the MelC1 and MelC2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
抗生素链霉菌的酪氨酸酶由黑色素操纵子（melC）的第二个开放阅读框melC2编码。上游开放阅读框melC1编码一种146个氨基酸的蛋白质，其具有分泌蛋白典型的NH2末端信号肽特征。MelC1蛋白通过二元复合物的形成参与将铜离子转移至脱辅基酪氨酸酶MelC2（Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71 - 81; Chen, L.-Y., Leu, W.-M., Wang, K.-T., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 20100 - 20107）。为研究酪氨酸酶的输出是否也依赖于MelC1，对其信号肽序列进行了突变研究。获得了四个不同的突变体。带正电荷区域的突变（突变体M - 6LE，Arg6 - Arg7----Leu6 - Glu7）或疏水区域的突变（突变体M - 16D，Val16----Asp16）导致Mel - 表型。这些损伤导致MelC1和MelC2蛋白的输出严重降低7至10倍，并使这两种蛋白在胞质部分积累。M - 6LE突变体中与细胞相关的酪氨酸酶活性显著降低至野生型菌株中活性的4%，而M - 16D突变体中则未降低，这表明MelC1的碱性NH2末端对该蛋白的反式激活功能也很重要。然而，突变体M - 6LE和M - 16D中MelC1的反式激活和/或分泌功能缺陷并非由于MelC1.MelC2复合物形成受损。这两个突变体中黑色素操纵子基因的翻译也减少。相反，如果突变发生在信号肽酶的假定切割位点（例如突变体M - 29SM，Arg29 - Ala30----Ser29 - Met30或突变体29 - SMG，Arg29 - Ala30 - Asp31----Ser29 - Med30 - Gly31+++），酪氨酸酶活性和MelC2的分泌不受影响。此外，MelC1阴性突变体M - 950中酪氨酸酶活性及其输出被消除。综上所述，这些结果表明功能性MelC1对于酪氨酸酶分泌和活性至关重要。此外，结果表明，与其他分泌蛋白一样，MelC1信号序列中的碱性和疏水残基是信号肽的重要特征，在MelC1和MelC2蛋白的分泌中起关键作用。（摘要截断于400字）
