The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells

To test the hypothesis that the SRIF propeptide mediates in-traceUular transport, we expressed two chimeric proteins in GH3 cells: PRO-GLO, a fusion of the SRIF preproregion and chimpanzee c~-globin; and a control precursor, SIG-GLO, consisting of the/3-1actamase signal peptide fused to ct-globin ( Fig. 1 B) . The rationale for these experiments was to determine if the propeptide could facilitate transport of a normally cytoplasmic protein through the secretory pathway. In addition, our aim was to determine if PRO-GLO would be proteolytically processed at the proSRIF ArgLys processing site and if the resulting globin polypeptide would be targeted to the regulated or constitutive secretory pathway. The starting cDNAs for these constructions were plasmids pSP125E encoding the/~-lactamase signal peptide fused to chimpanzee u-globin (40) , and pDWS18 which contained anglerfish preproSRIF I cDNA (13) cloned into the Eco RI site ofa pBR322 derivative in which all four Nae I sites were deleted (Fig. 1 ). After digestion, the appropriate DNA fragments were ligated into the Barn HI site of the retroviral expression vector pLJ (15) and were designated pLJSIG-GLO and pLJPRO-GLO, respectively. Plasmid pLJSIG-GLO encoded the/~-lactamase signal peptide (26 amino acids) fused to ct-globin (40) . Plasmid pLJPRO-GLO encoded the pre-proSRIF I signal peptide, 82 residue proregion, the first amino acid of mature SRIF (Ala), three residues (AspPro-Arg) resulting from the cloning and the complete chimpanzee a-globin chain sequence (Fig. 1 , PrePROSRIF-GLO). We used the expression vector pLJ since the recombinant virions can infect rat pituitary GH3 cells and these cells efficiently cleave native preproSRIF to the mature hormone (41) .