Tong et al. reveal that a zone of GTPase inhibition prevents a new bud site from overlapping with the previous site.
The process of budding leaves behind a scar in the cell wall. Each cell cycle creates a new yeast scar, as new division sites never fall on top of previous ones. Scientists wondered whether the scars might make the cell wall too rigid for a new bud site to form there. But the new findings show that the physical properties of the wall are not to blame.
New bud sites repeatedly formed at the same site—on top of a scar—when a GTPase-activating protein (GAP) called Rga1 was deleted. This GAP's target is Cdc42, a polarity-inducing Rho GTPase. In its GTP-bound form, Cdc42 points the cytoskeleton and thus vesicular traffic toward the new bud site. But Rga1 inactivates Cdc42 by inducing it to hydrolyze its GTP to GDP.
During and after cytokinesis in wild-type cells, Rga1 was concentrated between two rings of septins, which help separate the cells. Its presence prevented active Cdc42 from accumulating there. Cdc42-GTP instead formed a patch just outside the Rga1 ring, creating a new bud next to, but not on top of, the previous site.
Although other GAPs that inactivate Cdc42 localized between the septin rings, they could not substitute for Rga1 to prevent budding on top of scars. This sort of specialization might help explain why humans have 68 GAPs for just 17 Rho GTPases.
The Rga1 mutant cells survived in culture despite their overlapping bud sites. In the wild, however, the mutation would be disadvantageous; in nature, buds often remain attached to their mother cell and would thus obstruct subsequent buds. Yeast cells probably senesce before their surface becomes completely covered with scars.