The Ubiquitin Ligase SCFUcc1 Acts as a Metabolic Switch for the Glyoxylate Cycle

Nakatsukasa, Kunio; Nishimura, Takashi; Byrne, Stuart D.; Okamoto, Michiyo; Takahashi‐Nakaguchi, Azusa; Chibana, Hiroji; Okumura, Fumihiro; Kamura, Takumi

doi:10.1016/j.molcel.2015.04.013

Cited by 39 publications

(48 citation statements)

References 57 publications

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“…This suggests that a balance between the monomeric and dimeric forms of Cit1p may play a role in its regulation, although this remains to be proven. Second, the thioredoxin-mediated redox regulation of citrate synthase in Arabidopsis and the regulation of the glyoxylate cycle Cit2p by ubiquitinylation in yeast suggest that post-translational modification of citrate synthase can be beneficial (Nakatsukasa et al, 2015; Schmidtmann et al, 2014). Finally, acute regulation of citrate synthase could have cellular benefits.…”

Section: Discussionmentioning

confidence: 99%

Ptc7p Dephosphorylates Select Mitochondrial Proteins to Enhance Metabolic Function

et al. 2017

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SUMMARY Proper maintenance of mitochondrial activity is essential for metabolic homeostasis. Widespread phosphorylation of mitochondrial proteins may be an important element of this process; yet little is known about which enzymes control mitochondrial phosphorylation, or which phosphosites have functional impact. We investigate these issues by disrupting Ptc7p—a conserved but largely uncharacterized mitochondrial matrix PP2C-type phosphatase. Loss of Ptc7p causes respiratory growth defects concomitant with elevated phosphorylation of select matrix proteins. Among these, Δptc7 yeast exhibit an increase in phosphorylation of Cit1p—the canonical citrate synthase of the tricarboxylic acid cycle—that diminishes its activity. We find that phosphorylation of S462 can eliminate Cit1p enzymatic activity likely by disrupting its proper dimerization, and that Ptc7p-driven dephosphorylation rescues Cit1p activity. Collectively, our work connects Ptc7p to an essential TCA cycle function and to additional phosphorylation events that may affect mitochondrial activity inadvertently or in a regulatory manner.

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Section: Discussionmentioning

confidence: 99%

Ptc7p Dephosphorylates Select Mitochondrial Proteins to Enhance Metabolic Function

et al. 2017

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“…Degradation of ERAD substrates was analyzed by cycloheximide chase as described previously [ 46 , 48 ].…”

Section: Methodsmentioning

confidence: 99%

Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation

Nakatsukasa

Kamura

2016

PLoS ONE

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During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast. Consistent with previous in vitro studies, ubiquitinated Ste6* was extracted from P20 (20,000 g pellet) fraction to S20 (20,000 g supernatant) fraction in a Cdc48/p97-dependent manner. Similarly, Ubx2p, which recruits Cdc48/p97 to the ER, facilitated the extraction of Ste6*. By contrast, lipid droplet formation, which was suggested to be dispensable for the degradation of Hrd1-substrates in yeast, was not required for the degradation of Ste6*. Intriguingly, we found that ubiquitinated Ste6* in the S20 fraction could be enriched by further centrifugation at 100,000 g. Although it is currently uncertain whether ubiquitinated Ste6* in P100 fraction is completely free from any lipids, membrane flotation analysis suggested the existence of two distinct populations of ubiquitinated Ste6* with different states of membrane association. Together, these results imply that ubiquitinated Ste6* may be sequestered into a putative quality control sub-structure by Cdc48/p97. Fractionation assays developed in the present study provide a means to further dissect the ill-defined post-ubiquitination step during ERAD of polytopic membrane substrates.

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“…Polyclonal anti‐Spo12 antibodies were generated in rabbits against recombinant Spo12 in our laboratory . Mouse monoclonal antibodies against FLAG (M2) and Pgk1 (3‐phosphoglycerate kinase 1) were obtained from Sigma‐Aldrich and Invitrogen, respectively.…”

Section: Methodsmentioning

confidence: 99%

The HECT‐type ubiquitin ligase Tom1 contributes to the turnover of Spo12, a component of the FEAR network, in G2/M phase

et al. 2018

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The ubiquitin-proteasome system plays a crucial role in cell cycle progression. A previous study suggested that Spo12, a component of the Cdc14 early anaphase release (FEAR) network, is targeted for degradation by the APC/C complex in G1 phase. In the present study, we demonstrate that the Hect-type ubiquitin ligase Tom1 contributes to the turnover of Spo12 in G2/M phase. Coimmunoprecipitation analysis confirmed that Tom1 and Spo12 interact. Overexpression of Spo12 is cytotoxic in the absence of Tom1. Notably, Spo12 is degraded in S phase even in the absence of Tom1 and Cdh1, suggesting that an additional E3 ligase(s) also mediates Spo12 degradation. Together, we propose that several distinct degradation pathways control the level of Spo12 during the cell cycle.

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The Ubiquitin Ligase SCFUcc1 Acts as a Metabolic Switch for the Glyoxylate Cycle

Cited by 39 publications

References 57 publications

Ptc7p Dephosphorylates Select Mitochondrial Proteins to Enhance Metabolic Function

Ptc7p Dephosphorylates Select Mitochondrial Proteins to Enhance Metabolic Function

Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation

The HECT‐type ubiquitin ligase Tom1 contributes to the turnover of Spo12, a component of the FEAR network, in G2/M phase

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