Intracellular trafficking and metabolic turnover of yeast prepro-α-factor-SRIF precursors in GH3 cells

Abstract

Chimeric genes coding for prepro region of yeast α α α α-factor and anglerfish SRIF were expressed in rat GH 3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast α α α α-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that ~68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of ~2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.

Directed evolution of a secretory leader for the improved expression of heterologous proteins and full‐length antibodies in Saccharomyces cerevisiae

“…Commonly used as a secretory peptide (Brake, 1984;Kjaerulff and Jensen, 2005;Lee et al, 2002;Oka et al, 1999), the alpha mating factor 1 secretory leader (MFa1pp) is made of two distinct pieces: the pre region and the pro region. The first 19 residues make up the pre region that serves to direct the nascent polypeptide to the endoplasmic reticulum.…”

“…The MFa1pp is commonly used to direct the secretion of a number of different heterologous proteins in a number of hosts including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and mammalian cells, and its success as a secretory leader depends a great deal on the protein being directed (Kjaerulff and Jensen, 2005;Lee et al, 2002;Oka et al, 1999). Secretory leaders have previously been engineered by iterative processes of rational design and empirical optimization, specifically for the secretion of insulin precursor.…”

The cell toxicity effect of secalonic acid D on GH3 cells and the related mechanisms

“…SAD may also affect organelles such as mitochondria, ribosome and endoplasmic reticulum, for pituitary adenoma cells have activity in synthesis and secretion of many kinds of hormones and abundance of organelles. It is reported that many important factors that regulate the expression of GH were synthesized and secreted in endoplasmic reticulum and Golgi such as Somatotropin release inhibiting factor (SRIF) (33), vascular endothelial growth factor (VEGF) (34), carboxypeptidase D (CPD) (35). SAD may indirectly influence these factors to reduce the expression of GH.…”

Use of a protein engineering strategy to overcome limitations in the production of “Difficult to Express” recombinant proteins

“…Synthetic signal peptides and leader sequences have been used extensively in yeast expression systems to enhance heterogeneous protein production (Brake et al, ; Heiss et al, ; Hou et al, ; Rakestraw et al, ). Use of the yeast mutant alpha mating factor 1 leader peptide (MFα1pp) in mammalian cells (rat pituitary‐derived cell line) resulted in efficient ER‐Golgi translocation of a neuropeptide (somatostatin), but led to inefficient processing and intracellular accumulation during later stages of the secretory pathway (Lee et al, ).…”