%0 Journal Article %A Heiss, Silvia %A Puxbaum, Verena %A Gruber, Clemens %A Altmann, Friedrich %A Mattanovich, Diethard %A Gasser, Brigitte %T Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization %D 2015 %J Microbiology, %V 161 %N 7 %P 1356-1368 %@ 1465-2080 %R https://doi.org/10.1099/mic.0.000105 %I Microbiology Society, %X Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport. %U https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000105