Abstract
In our previous study, we found that Ypt1p, a Rab family small GTPase protein, exhibits a stress-driven structural and functional switch from a GTPase to a molecular chaperone, and mediates thermo tolerance in Saccharomyces cerevisiae. In the current study, we focused on the temperature-sensitive ypt1-G80D mutant, and found that the mutant cells are highly sensitive to heat-shock, due to a deficiency in the chaperone function of Ypt1pG80D. This defect results from an inability of the protein to form high molecular weight polymers, even though it retains almost normal GTPase function. The heat-stress sensitivity of ypt1-G80D cells was partially recovered by treatment with 4-phenylbutyric acid, a chemical chaperone. These findings indicate that loss of the chaperone function of Ypt1pG80D underlies the heat sensitivity of ypt1-G80D cells. We also compared the proteomes of YPT1 (wild-type) and ypt1-G80D cells to investigate Ypt1p-controlled proteins under heat-stress conditions. Our findings suggest that Ypt1p controls an abundance of proteins involved in metabolism, protein synthesis, cellular energy generation, stress response, and DNA regulation. Finally, we suggest that Ypt1p essentially regulates fundamental cellular processes under heat-stress conditions by acting as a molecular chaperone.
Highlights
Ypt1p is a member of the Rab family of small GTPases that cycles between an active GTP-bound form and an inactive GDP-bound form
Hydrolysis of Ypt1p-bound GTP is an essential step in vesicle transport and cell growth, yet the GTPase-deficient ypt1-Q67L mutant exhibits no observable defects in protein transport, secretion, membrane morphology, or cell growth at temperatures ranging from 14 ◦C to 37 ◦C [4]
We found that heat-shock induces a functional switch in Ypt1p from that of a GTPase to that of a molecular chaperone, and this change is driven by a structural switch from a low molecular weight (LMW) to a high molecular weight (HMW) form
Summary
Ypt1p is a member of the Rab family of small GTPases that cycles between an active GTP-bound form and an inactive GDP-bound form. Hydrolysis of Ypt1p-bound GTP is an essential step in vesicle transport and cell growth, yet the GTPase-deficient ypt1-Q67L mutant exhibits no observable defects in protein transport, secretion, membrane morphology, or cell growth at temperatures ranging from 14 ◦C to 37 ◦C [4] Based on these findings, it was concluded that, contrary to the general concept of Ypt1p/Rab function, the GTPase activity of Ypt1p is not essential for Ypt1p-mediated vesicle transport or membrane fusion, or for growth at an elevated temperature (37 ◦C). The Ypt1pG80D protein has normal GTPase function and the ypt1-G80D mutant strain displays normal growth and nearly normal endoplasmic reticulum-to-Golgi vesicle trafficking at typical growth temperature (30 ◦C), but experiences growth retardation at an elevated temperature (37 ◦C) [7] This finding implies that the GTPase activity of Ypt1p is not essential for the growth of yeast at elevated temperatures
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