Delivery of a secreted soluble protein to the vacuole via a membrane anchor

To further understand how membrane proteins are sorted in the secretory sys tem, we devised a strategy that involves the expression of a membrane-ancho red yeast invertase in transgenic plants. The construct consisted of a sign al peptide followed by the coding region of yeast invertase and the transme mbrane domain and cytoplasmic tail of calnexin. The substitution of a lysin e near the C terminus of calnexin with a glutamic acid residue ensured prog ression through the secretory system rather than retention in or return to the endoplasmic reticulum. In the transformed plants, invertase activity an d a 70-kD cross-reacting protein were found in the vacuoles. This yeast inv ertase had plant-specific complex glycans, indicating that transport to the vacuole was mediated by the Golgi apparatus. The microsomal fraction conta ined a membrane-anchored 90-kD cross-reacting polypeptide, but was devoid o f invertase activity. Our results indicate that this membrane-anchored prot ein proceeds in the secretory system beyond the point where soluble protein s are sorted for secretion, and is detached from its membrane anchor either just before or just after delivery to the vacuole.