Tonoplast and Soluble Vacuolar Proteins Are Targeted by Different Mechanisms

The delivery of proteins to the vacuole and its limiting membrane (the tonoplast) by the secretory system is thought to be a dissociative process in which vesicles bud from one compartment and fuse with another. We studied the transport kinetics of phytohemagglutinin (PHA) and tonoplast intrinsic protein (TIP) in mesophyll protoplasts obtained from transgenic tobacco plants transformed with genes encoding these two proteins. In pulse-chase experiments, arrival of PHA in the vacuole was found to be slower (completed 24 hr after synthesis) than the arrival of TIP in the tonoplast (completed 6 hr after synthesis). Brefeldin A and monensin block protein transport by interfering in specific vesicle transport steps. Brefeldin A prevents anterograde vesicle transport between the endoplasmic reticulum and the Golgi, whereas monensin inhibits correct sorting in the trans-Golgi network by disrupting the proton gradient across the membrane. Both inhibitors blocked the transport of PHA to the vacuole and altered the rate at which its complex glycan is processed by Golgi enzymes. Neither drug stopped the arrival of TIP in the tonoplast, suggesting that the flow of vesicles continues in the presence of these inhibitors. We suggest that soluble proteins like PHA and membrane proteins like TIP reach their vacuolar destinations by different paths.