Switching of cargo sorting from the constitutive to regulated secretory pathway by the addition of cystatin D sequence in salivary acinar cells.

The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the β-adrenergic receptor agonist isoproterenol, as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, while fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi network. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.