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. 2016 Apr;100(2):303-14.
doi: 10.1111/mmi.13319. Epub 2016 Mar 10.

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae

Kejin Hu  1 , Shuguang Guo  2 , Gonghong Yan  1 , Wenjie Yuan  3 , Yin Zheng  4 , Yu Jiang  1
Affiliations

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae

Kejin Hu et al. Mol Microbiol. 2016 Apr.

Abstract

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth-related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12-rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2(W2041R) ) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non-covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization.

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Figures

Figure 1.
Figure 1.. The tor2–2041 mutant is temperature sensitive and rapamycin resistant.
A. Temperature sensitive and rapamycin resistant traits of the tor2–2041 mutant. Wild type TOR2 control (Y818), tor1 tor2–2041 (Y812), and tor1 TOR2S1975R (Y031) mutant cells were placed on either YPD or YPD plate containing 100 nM of rapamycin. The plates were imaged after incubated for three days at either 23 or 37°C. B. The tor2–2041 mutant is defective for both the TOR-shared and TOR2-unique function. Wild type TOR2 (Y818), TOR1 tor2–2041 (Y817), and tor1 tor2–2041 (Y812) cells were placed on either YPD or YPD plate containing 1 M sorbitol. The plates were imaged after incubated for three days at either 23 or 37°C.
Figure 2.
Figure 2.. The W2041 residue is located within the rapamycin binding pocket of the FRB domain.
A. Sequence alignment of the FRB domains in Tor proteins. Δ indicates the conserved S1975 in Tor2 that confers yeast rapamycin resistant, but not temperature sensitive. ∗ designates the conserved W2041 in Tor2 that, when mutated to arginine, renders yeast rapamycin resistant and temperature sensitive. Other aromatic residues that contribute to rapamycin binding are underscored by #. dTOR: drosophila Tor. B. 3D structure of the FRB domain in complex with FKBP12 and rapamycin (PDB ID: 1FAP) with superimposed positions of W2041 and S1975.
Figure 3.
Figure 3.. The tor2–2041 mutant is defective for TORC1 function.
A. Exponentially growing wild type (Y818) and tor2 mutant (Y812 and Y817) cells expressing GFP-ATG8 were incubated at 23 and 37°C for indicated times or treated with 100 nM rapamycin for 1 hr at 23°C (Rap). The cell extracts were analyzed by western blotting with anti-GFP antibody for the autophagic cleavage of GFP-Atg8 fusion protein. B. Exponentially growing yeast wild type TOR2 (Y110) and mutant tor2–2041 (Y1035) cells expressing HA tagged wild type or mutant Tor2 protein were incubated at 23 and 37°C for indicated times. The expression levels of the HA tagged Tor2 protein and the loading control (Tpd3) was examined by western blotting. C. Cell extracts from exponentially growing wild type (Y110) and mutant tor2–2041 (Y1035) expressing KOG1-myc cells were precipitated with anti-HA antibody. The levels of HA-Tor2 and Kog1-myc in the extract and precipitates were determined by western blotting. D. Wild type TOR2 (Y110) and mutant tor2–2041 (Y1035) expressing control vector, KOG1-HA or AVO1-HA were shifted from 23 to 37°C for 2 hr. Cell extracts from cells before and after the shift were fractionated into pellet (P) and soluble fractions (S) by centrifugation at 100, 000 ×g for 1 h. The levels of HA-Tor2, Kog1-HA, or Avo1-HA in the soluble and pellet fraction were examined by western blotting. E. Wild type TOR2 (Y818) TOR1 tor2–2041 (Y817), and tor1 tor2–2041 (Y812) cells were shifted from 23 to 37°C. The levels of Kog1-HA in the cells collected at different time points after the shift were examined by western blotting. F. Wild type cells (Y662) were transformed with control vector (lane 1) or KOG1-myc (lanes 2–4) together with either HA-TOR2 (lanes 1–3) or HA-tor2–2041(lanes 4–5). Transformed cells were grown at 23°C and shifted to 37°C for 2 hr. Cell extracts were precipitated with anti-HA antibody and the levels of HA-Tor2 and Kog1-myc in the extracts (Ext) and precipitates (HAIP) were examined by western blotting. G. Wild type cells (Y662) were transformed with control vector (lane 1) or AVO1-myc (lanes 2–4) together with either HA-TOR2 (lanes 1–3) or HA-tor2–2041(lanes 4–5). Transformed cells were grown at 23°C and shifted to 37°C for 2 hr. Cell extracts were precipitated with anti-HA antibody and the levels of HA-Tor2 and Avo1-myc in the extracts (Ext) and precipitates (HA-IP) were examined by western blotting.
Figure 4.
Figure 4.. Overexpression of ubiquitin suppresses the growth defect of the tor2–2041 mutant.
The tor2–2041 cells (Y864) were transformed with control vector, or vectors expressing ubiquitin or wild type TOR2. The transformants were plated on SC-URA plates and imaged after incubation at 23 or 37°C for three days.
Figure 5.
Figure 5.. Overexpression of ubiquitin prevents Kog1 degradation in the tor2–2041 mutant cells.
The tor1 tor2–2041 (Y864) cells expressing KOG1-HA were transformed with empty vector or vector expressing ubiquitin. The transformed cells were grown to exponential phase at 23°C and shifted to 37°C or remained at 23°C. A. The levels of Kog1-HA in the cell extracts from the cells collected at different time points after the shift were determined by western blotting. Wild type cells expressing KOG1-HA (Y985) were used as control. The levels of Tpd3 were used as the loading control. B. Cell extracts were partitioned into membrane (P) and soluble (S) by centrifugation at 100, 000 × g. The distribution of Kog1-HA in each fraction was examined by western blotting. C. The tor1 tor2–2041 (Y864) cells were transformed with the GFP-ATG8 gene together with a control vector or vector expressing ubiquitin. The transformed cells were grown to exponential phase at 23°C and shifted to 37°C for indicated times. The cell extracts were analyzed by western blotting with anti-GFP antibody for the autophagic cleavage of GFP-Atg8 fusion protein.
Figure 6.
Figure 6.. Ubiquitination is not involved in the effect of ubiquitin in suppression of the growth defect of the tor2–2041 mutant.
Exponentially growing tor1 tor2–2041 (Y864) cells expressing wild type or mutant ubiquitin were spotted on SC-TRP plates after a series of 10-fold dilutions. Plates were imaged after incubation at 23 or 37°C for 4 days.
Figure 7.
Figure 7.. Ubiquitin binds to Kog1 in response to temperature upshift.
A. Wild type yeast cells expressing KOG1-HA (Y985) and ubiquitin were subjected to heat stress for 1 hr. Cell extracts were precipitated with anti-HA antibody and the levels of Kog1-HA and ubiquitin in the cell extracts and precipitates were analyzed by western blotting. B. Wild type yeast cells expressing KOG1-HA (Y985), together with myc-tagged ubiquitin or ubiquitin G76A mutant were subjected to heat stress for 1 hr. Cell extracts were precipitated with anti-HA antibody and the levels of Kog1-HA and myc tagged ubiquitin in the extracts and precipitates were examined by western blotting.
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