Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae.

TLDR

Sec4 is a ras‑family GTP‑binding protein essential for exocytosis in Saccharomyces cerevisiae. The authors aimed to determine the role of GTP hydrolysis in Sec4 function by creating a Q‑79‑L mutation analogous to Ras Q‑61‑L in a region that contacts the GTP phosphoryl group. They constructed the Q‑79‑L mutant and assessed its intrinsic hydrolysis, GAP‑stimulated activity, and cellular phenotypes. The Q‑79‑L mutation eliminates intrinsic GTP hydrolysis, is only partially rescued by a GAP to 30 % of wild‑type activity, causes accumulation of GTP‑bound Sec4, supports viability but induces cold‑sensitivity, impaired invertase secretion, vesicle accumulation, and synthetic lethality, demonstrating that Sec4’s GTP/GDP cycling is essential for vesicular transport and distinct from Ras.

Abstract

Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.