Interphase chromosome condensation in nutrient-starved conditions requires Cdc14 and Hmo1, but not condensin, in yeast.

When asynchronously growing cells suffer from nutrient depletion and inactivation of target of rapamycin complex 1 (TORC1) protein kinase, the rDNA (rRNA gene) region is condensed in budding yeast Saccharomyces cerevisiae, which is executed by condensin and Cdc14 protein phosphatase. However, it is unknown whether these mitotic factors can condense the rDNA region in nutrient-starved interphase cells. Here, we show that condensin is not involved in TORC1 inactivation-induced rDNA condensation in G1 cells. Instead, the high-mobility group protein Hmo1 drove this process. The histone deacetylase Rpd3 and Cdc14, which repress rRNA transcription, were both required for the interphase rDNA condensation. Furthermore, interphase rDNA condensation necessitated CLIP and cohibin that tether rDNA to inner nuclear membranes. Finally, we showed that Hmo1, CLIP, Rpd3, and Cdc14 were required for survival in nutrient-starved G1 cells. Thus, this study disclosed novel features of interphase chromosome condensation.

TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells.

Unsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually override mitotic arrest by mitotic slippage. Here, we show that inactivation of TORC1 kinase elicits mitotic slippage in budding yeast and human cells. Yeast mitotic slippage was accompanied with aberrant aspects, such as degradation of the nucleolar protein Net1, release of phosphatase Cdc14, and anaphase-promoting complex/cyclosome (APC/C)-Cdh1-dependent degradation of securin and cyclin B in metaphase. This mitotic slippage caused chromosome instability. In human cells, mammalian TORC1 (mTORC1) inactivation also invoked mitotic slippage, indicating that TORC1 inactivation-induced mitotic slippage is conserved from yeast to mammalian cells. However, the invoked mitotic slippage in human cells was not dependent on APC/C-Cdh1. This study revealed an unexpected involvement of TORC1 in mitosis and provides information on undesirable side effects of the use of TORC1 inhibitors as immunosuppressants and anti-tumor drugs.

The PI3 Kinase Complex II-PI3P-Vps27 Axis on Vacuolar Membranes is Critical for Microautophagy Induction and Nutrient Stress Adaptation.

Phosphatidylinositol 3-phosphate (PI3P), a scaffold of membrane-associated proteins required for diverse cellular events, is produced by Vps34-containing phosphatidylinositol 3-kinase (PI3K). PI3K complex I (PI3KCI)-generated PI3P is required for macroautophagy, whereas PI3K complex II (PI3KCII)-generated PI3P is required for endosomal sorting complex required for transport (ESCRT)-mediated multi-vesicular body (MVB) formation in late endosomes. ESCRT also promotes vacuolar membrane remodeling in microautophagy after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Whereas PI3KCI and macroautophagy are critical for the nutrient starvation response, the physiological roles of PI3KCII and microautophagy during starvation are largely unknown. Here, we showed that PI3KCII-produced PI3P on vacuolar membranes is required for microautophagy induction and survival in nutrient-stressed conditions. PI3KCII is required for Vps27 (an ESCRT-0 component) recruitment and ESCRT-0 complex formation on vacuolar surfaces after TORC1 inactivation. Forced recruitment of Vps27 onto vacuolar membranes rescued the defect in microautophagy induction in PI3KCII-deficient cells, indicating that a critical role of PI3P on microautophagy induction is Vps27 recruitment onto vacuolar surfaces. Finally, vacuolar membrane-associated Vps27 was able to recover survival during nutrient starvation in cells lacking PI3KCII or Vps27. This study revealed that the PI3KCII-PI3P-Vps27 axis on vacuolar membranes is critical for ESCRT-mediated microautophagy induction and nutrient stress adaptation.

Cdc14 phosphatase downmodulates ESCRT-0 complex formation on vacuolar membranes and microautophagy after TORC1 inactivation.

Remodeling of vacuolar membranes mediated by endosomal sorting complex required for transport (ESCRT) is critical for microautophagy induction in budding yeast. Nutrient depletion and inactivation of target of rapamycin complex 1 (TORC1) protein kinase elicit recruitment of the ESCRT-0 complex (Vps27-Hse1) onto vacuolar membranes and ESCRT-mediated microautophagy induction. Mitotic protein phosphatase Cdc14 antagonizes TORC1-mediated phosphorylation in macroautophagy induction after nutrient starvation and TORC1 inactivation. Here, we report that Cdc14 downregulates microautophagy induction after TORC1 inactivation. Cdc14 dysfunction stimulated the vacuolar membrane recruitment of Hse1, but not Vps27, after TORC1 inactivation, promoting ESCRT-0 complex formation. Conversely, overexpression of CDC14 compromises Hse1 recruitment on vacuolar membranes and microautophagy induction after TORC1 inactivation. Thus, Cdc14 phosphatase regulates the fluxes of two types of autophagy in the opposite directions, namely, it elicits macroautophagy and attenuates microautophagy.

The vacuole controls nucleolar dynamics and micronucleophagy via the NVJ.

Chromosomes have their own territories and dynamically translocate in response to internal and external cues. However, whether and how territories and the relocation of chromosomes are controlled by other intracellular organelles remains unknown. Upon nutrient starvation and target of rapamycin complex 1 (TORC1) inactivation, micronucleophagy, which preferentially degrades nucleolar proteins, occurs at the nucleus-vacuole junction (NVJ) in budding yeast. Ribosomal DNA (rDNA) is condensed and relocated against the NVJ, whereas nucleolar proteins move towards the NVJ for micronucleophagic degradation, causing dissociation of nucleolar proteins from rDNA. These findings imply that the NVJ is the critical platform in the directional movements of rDNA and nucleolar proteins. Here, we show that cells lacking the NVJ (NVJΔ cells) largely lost rDNA condensation and rDNA-nucleolar protein separation after TORC1 inactivation. The macronucleophagy receptor Atg39, an outer nuclear membrane protein, accumulated at the NVJ and was degraded by micronucleophagy. These suggested that macronucleophagy is also dependent on the presence of the NVJ. However, micronucleophagy, but not macronucleophagy, was abolished in NVJΔ cells. This study clearly demonstrated that vacuoles controls intranuclear events, nucleolar dynamics, from outside of the nucleus via the NVJ under the control of TORC1.

ESCRT machinery plays a role in microautophagy in yeast.

BACKGROUND: Microautophagy, which degrades cargos by direct lysosomal/vacuolar engulfment of cytoplasmic cargos, is promoted after nutrient starvation and the inactivation of target of rapamycin complex 1 (TORC1) protein kinase. In budding yeast, microautophagy has been commonly assessed using processing assays with green fluorescent protein (GFP)-tagged vacuolar membrane proteins, such as Vph1 and Pho8. The endosomal sorting complex required for transport (ESCRT) system is proposed to be required for microautophagy, because degradation of vacuolar membrane protein Vph1 was compromised in ESCRT-defective mutants. However, ESCRT is also critical for the vacuolar sorting of most vacuolar proteins, and hence reexamination of the involvement of ESCRT in microautophagic processes is required. RESULTS: Here, we show that the Vph1-GFP processing assay is unsuitable for estimating the involvement of ESCRT in microautophagy, because Vph1-GFP accumulated highly in the prevacuolar class E compartment in ESCRT mutants. In contrast, GFP-Pho8 and Sna4-GFP destined for vacuolar membranes via an alternative adaptor protein-3 (AP-3) pathway, were properly localized on vacuolar membranes in ESCRT-deficient cells. Nevertheless, microautophagic degradation of GFP-Pho8 and Sna4-GFP after TORC1 inactivation was hindered in ESCRT mutants, indicating that ESCRT is indeed required for microautophagy after nutrient starvation and TORC1 inactivation. CONCLUSIONS: These findings provide evidence for the direct role of ESCRT in microautophagy induction.

TORC1 regulates G1/S transition and cell proliferation via the E2F homologs MBF and SBF in yeast.

The yeast E2F functional homologs MBF (Mbp1/Swi6) and SBF (Swi4/Swi6) complexes are critical transcription factors for G1/S transition. The target of rapamycin complex 1 (TORC1) kinase promotes G1/S transition via upregulation of the G1 cyclin Cln3 that activates MBF and SBF in favorable nutrient conditions. Here, we show evidence that TORC1 directly regulates G1/S transition via MBF and SBF. Various proteins involved in G1/S transition, including Mbp1 and Swi4, but not Swi6, were largely lost after rapamycin treatment. TORC1 inactivation facilitated degradation of Mbp1 and Swi4. Mbp1 degradation was dependent on Skp1-Cullin1-F-box (SCF)-Grr1 and proteasomes. We identified a PEST-like degron in Mbp1. Mutant cells with an unstable Mbp1 protein were hypersensitive to rapamycin and more accumulated G1 cells in the absence and presence of rapamycin. This study revealed that TORC1 directly controls MBF/SBF-mediated G1/S transition in response to nutrient availability.