Effects of mutations within and flanking the bovine growth hormone signal peptide on protein secretion

The mechanism of protein secretion involves a complicated series of events involving signal peptide recognition, translocation across several cellular membranes, molecular chaperonin-mediated protein folding and conformation-dependent processivity through the secretory pathway. Much of our understanding of eukaryotic protein secretion at the molecular level has been elucidated from in vitro studies or from genetic studies performed in bacteria or yeast. This study describes a mutational analyses of protein secretion using a normally exported protein, bovine growth hormone (bGH), as reporter. Following transfection of cultured fibroblasts, a transient gene expression system was employed to assess the effects of a battery of oligonucleotide-directed bGH signal peptide mutations on bGH secretion by intact cells.;BGH signal peptide mutations were directed to the signal peptidase cleavage domain or to the hydrophobic core of the bGH signal peptide. Furthermore, the effects of inserting an endoplasmic reticulum retention signal (KDEL) at the COOH terminus of bGH or deleting specific bGH exon domains were determined using assays which characterize secretion efficiency.;Radioimmunoassay and immunoprecipitation analyses revealed a gradient of secretion defects corresponding to specific amino acid substitutions or deletions. The singular effect of mutations which flank the signal peptidase processing domain demonstrated a coupling between processing and translocation, resulting in either altered cleavage specificity and/or decreased processing kinetics. Evidence supporting this tenet was demonstrated by characterization of the Val:Glu;In vitro receptor binding was performed with murine microsomal membranes and either bGH-;Several mutated bGH molecules were assessed for in vivo growth promotion by producing transgenic mice. Expression of bGH-...