When yeast cells overproduce the Rab blocker Gyp7, their vacuoles fragment (right).

It takes more than one inhibitor to keep a Rab down, Brett et al. report. The team determined that yeast cells use two switches to turn off this vesicle management protein.

The Rab proteins regulate the transport and fusion of vesicles. Researchers have teased out the Rab control pathways in vitro, discovering that GTPase-activating proteins (GAPs) turn off Rabs. But scientists know little about what happens in living cells.

So Brett et al. followed the dynamics of yeast vacuoles, which split or fuse depending on the cell's situation. To nail down what inactivates a fusion-promoting Rab called Ypt7, the scientists cranked up the levels of different Gyp proteins—the yeast versions of GAPs. Confirming results of in vitro studies, the researchers showed that Gyp7 inhibits Ypt7.

But Gyp7 can't do the job alone, the team found. It needs help from another protein called Yck3, which phosphorylates two targets of Ypt7: a protein complex that tethers vacuoles to each other and a second complex that promotes vacuole fusion. Ypt7 doesn't take this interference lying down, however. The researchers determined that active Ypt7 blocks phosphate addition.

The study helps fill in the complex control circuit for Ypt7. The team proposes that the network includes a “feed forward” loop, in which Ypt7 maintains its own activation by pre-empting Yck3. The end result might be a signaling circuit that is particularly sensitive to changes in Ypt7 activity.

Brett, C.L., et al.
J. Cell Biol.