Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.Over the years, heterologous protein expression in yeast species such as Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe has become an important tool in the production of therapeutic and commercially relevant proteins. The advantage of a yeast-based expression system is due to the fact that as a eukaryotic microorganism, yeast combines ease of genetic manipulation and cell growth with a well-known capacity to perform complex posttranslational protein modifications (4,22,29). Because yeast per se secretes only low levels of endogenous proteins, a secreted recombinant protein usually contributes significantly to the total amount of proteins in the culture medium. Thus, directing a foreign protein for secretion is an important initial step in its subsequent purification.While the vast majority of heterologous proteins has been expressed within the cytosol of the corresponding host, only a few proteins have been successfully secreted into the extracellular medium. In most eukaryotic proteins, the critical initial step in protein secretion is their co-and/or posttranslational translocation into the lumen of the endoplasmic reticulum (ER) followed by subsequent sorting into the Golgi network. Foreign protein import into the ER is usually achieved by fusing the protein of interest in frame to a homologous secretion signal sequence derived from a naturally secreted protein of the corresponding host, thus conferring secretion competence to the desired protein fusion (13,23).In yeast, the most widely used secretion sign...
