CLIP and cohibin separate rDNA from nucleolar proteins destined for degradation by nucleophagy

Abstract

Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) in budding yeast induces nucleophagy, a selective autophagy process that preferentially degrades nucleolar components. DNA, including ribosomal DNA (rDNA), is not degraded by nucleophagy, even though rDNA is embedded in the nucleolus. Here, we show that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites. Nucleolar proteins move to sites proximal to the nuclear-vacuolar junction (NVJ), where micronucleophagy (or piecemeal microautophagy of the nucleus) occurs, whereas rDNA dissociates from nucleolar proteins and moves to sites distal to NVJs. CLIP and cohibin, which tether rDNA to the inner nuclear membrane, were required for repositioning of nucleolar proteins and rDNA, as well as effective nucleophagic degradation of the nucleolar proteins. Furthermore, micronucleophagy itself was necessary for the repositioning of rDNA and nucleolar proteins. However, rDNA escaped from nucleophagic degradation in CLIP- or cohibin-deficient cells. This study reveals that rDNA-nucleolar protein separation is important for the nucleophagic degradation of nucleolar proteins.

rDNA Condensation Promotes rDNA Separation from Nucleolar Proteins Degraded for Nucleophagy after TORC1 Inactivation

“…Condensin, Hmo1, and Rpd3-Sin3 Are Critical for Survival during Nutrient Starvation Loss of Atg39, Nvj1, CLIP, or cohibin leads to a rapid loss of cell viability during nitrogen starvation, which is similar to that found in autophagy-defective mutants (Mochida et al, 2015;Mostofa et al, 2018;Tsukada and Ohsumi, 1993). This fact suggested that nucleophagy and proper degradation of nucleolar proteins after nutrient starvation and TORC1 inactivation are critical for cell survival during nutrient starvation.…”

“…By contrast, the displacement of rDNA from the NVJ-proximal site does not seem to be essential for the escape of rDNA from nucleophagic degradation, because rDNA was intact after rapamycin treatment in CLIP and cohibin mutants despite impaired displacement of rDNA . To repeat this estimation, chromosome stability after rapamycin treatment in condensin, Hmo1, and Rpd3-Sin3 HDAC mutants was determined by pulsed-field gel electrophoresis (PFGE) analysis (Kobayashi et al, 2004;Mostofa et al, 2018). In S. cerevisiae, 150 rDNA repeats are tandemly arranged on chromosome XII (chr.…”

“…Nucleolar shrinkage might result from not only rDNA compaction but also the degradation of nucleolar proteins. In the budding yeast Saccharomyces cerevisiae, nucleophagy (autophagy for degradation of the nucleus) degrades a non-essential portion of the nucleus, nucleolar proteins, after nutrient starvation and TORC1 inactivation (Kvam and Goldfarb, 2007;Mochida et al, 2015;Mostofa et al, 2018;Rahman et al, 2018;Roberts et al, 2003). In macronucleophagy, autophagosomes sequester nucleolar proteins and subsequently fuse with lysosomes or vacuoles for degradation.…”

“…Similar to other autophagic processes, macronucleophagy and micronucleophagy are both induced by nutrient starvation and TORC1 inactivation (Mochida et al, 2015;Roberts et al, 2003). Yeast cells lacking Atg39 or Nvj1 cannot survive in nutrientstarved conditions (Mochida et al, 2015;Mostofa et al, 2018). This suggests that nucleophagy (macro-and micronucleophagy) is critical for survival in such conditions, although its physiological importance for survival remains elusive at present.…”

An ESCRT-LEM domain inner nuclear membrane protein surveillance system is poised to directly monitor the integrity of the nuclear envelope barrier and nuclear transport system

“…Additionally, the size of the necks at the base of the INM evaginations and nuclear envelope herniations is suggestive of the presence of a spiraling polymer analogous to those observed in vitro and in vivo (McCullough et al, 2018), supporting that these necks are likely stabilized by ESCRTs. Thus, while we acknowledge that such INM evaginations might not be a physiological event in the nuclear envelope sealing process, it remains tempting to speculate that they might be in the context of proposed mechanisms of nuclear egress be it Mega-RNPs (Speese et al, 2012;Jokhi et al, 2013), viruses (Lee et al, 2012(Lee et al, , 2016Arii et al, 2018), or in nucleophagy mechanisms (Roberts et al, 2003;Dou et al, 2015;Mochida et al, 2015;Mostofa et al, 2018) that so-far remain obscure but would require a membrane scission step. Interestingly, recent work suggests that herpes virus nuclear egress requires ESCRTs (Arii et al, 2018), including a role in controlling interesting INM extensions into the nucleus; such intranuclear membrane might also be relevant in cell types that have so-called "nucleoplasmic reticulum" .…”