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The role of the yeast pheromone prosegment--alpha-factor--in the export of the heterologous protein, the human growth hormone (hGH), in the culture medium of S. cerevisiae has been studied. Using genetic engineering constructions, it has been shown that different N-terminal signal peptides (SP) are not able to provide the hGH export. Transformant cells carrying the plasmid with a hybrid sequence encoding the prosegment-hGH (without SP) accumulate non-glycosylated pro-hGH in the cytosol. Only the combination of SP and the prosegment as the N-terminal fragment of the hGH precursor results in the processing and export of the mature form of the hormone. The origin (or type) of SP is of no significance. The glycosylation inhibitors--2-deoxy-D-glucose and tunicamycin--suppress the export but not the entry of hGH into the periplasm, thus indicating a critical role of the intactness of the prosegment polymannose chains for the efficient export of heterologous protein. A conclusion is drawn that the two preprosegment parts play different roles. The SP constituent of prepro-hGH introduces pro-GH into the general secretory pathway, while the prosegment resulting from the SP cleavage directs hGH from the cell into the culture medium.

Two Saccharomyces cerevisiae strains containing integrated copies of the MAT alpha 1 gene fused to the PHO5 promoter have been obtained by transformation of a MAT alpha 1 mutant. The strains differ in length of 5'-uncoding region of the MAT alpha 1 in the integrated constructions. The mating activity and the ability of these strains to express the yeast MF alpha 1 gene and the hyman alpha-N-interferon gene under the control of MF alpha 1 promoter was shown to be regulated by the exogenous inorganic phosphate. The level of intracellular alpha-N-interferon synthesized in these strains was several fold higher as compared with the wild type alpha mating type strain. At the same time the observed increase in intracellular production is not accompanied by an increase in the level of secreted alpha-N-interferon. On the contrary, one of the strains had a two-fold reduction in the rate of secretion.