Secretion of the Streptomyces tyrosinase is mediated through its trans- activator protein, MelC1

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-Met³⁰-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. These results also establish a novel role for MelC1 on tyrosinase transport in addition to a role in copper transfer to tyrosinase.