Analysis of the sorting sequences of vacuolar serine proteases, Isp6 and Psp3, in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe possesses two vacuolar serine proteases, Isp6 and Psp3, homologous to the budding yeast protease Prb1. Although both proteases are required for maturation of vacuolar enzymes, their transport mechanisms remain unclear. Here, we examined the vacuolar targeting pathways of Isp6 and Psp3. Fluorescence microscopy of C-terminal EGFP fusions showed that both proteins localized to the vacuolar lumen in wild-type cells. In contrast, vacuolar localization was lost in a vps34Δ strain but was unaffected in cells lacking Vps10, the CPY receptor. These results indicate that Isp6 and Psp3 are transported through the VPS pathway in a Vps10-independent manner. Further mutational analyses identified short internal regions (amino acids 117-125 in Psp3 and 131-139 in Isp6) that are essential for vacuolar targeting. Our findings show that both proteases contain specific internal determinants required for delivery to the vacuole.