Ubiquitination Occurs in the Mitochondrial Matrix by Eclipsed Targeted Components of the Ubiquitination Machinery

Ubiquitination is a critical type of post-translational modification in eukaryotic cells. It is involved in regulating nearly all cellular processes in the cytosol and nucleus. Mitochondria, known as the metabolism heart of the cell, are organelles that evolved from bacteria. Using the subcellular compartment-dependent α-complementation, we detect multiple components of ubiquitination machinery as being eclipsed distributed to yeast mitochondria. Ubiquitin conjugates and mono-ubiquitin can be detected in lysates of isolated mitochondria from cells expressing HA-Ub and treated with trypsin. By expressing MTS (mitochondrial targeting sequence) targeted HA-tagged ubiquitin, we demonstrate that certain ubiquitination events specifically occur in yeast mitochondria and are independent of proteasome activity. Importantly, we show that the E2 Rad6 affects the pattern of protein ubiquitination in mitochondria and provides an in vivo assay for its activity in the matrix of the organelle. This study shows that ubiquitination occurs in the mitochondrial matrix by eclipsed targeted components of the ubiquitin machinery, providing a new perspective on mitochondrial and ubiquitination research

Inactive Proteasomes Routed to Autophagic Turnover Are Confined within the Soluble Fraction of the Cell

Previous studies demonstrated that dysfunctional yeast proteasomes accumulate in the insoluble protein deposit (IPOD), described as the final deposition site for amyloidogenic insoluble proteins and that this compartment also mediates proteasome ubiquitination, a prerequisite for their targeted autophagy (proteaphagy). Here, we examined the solubility state of proteasomes subjected to autophagy as a result of their inactivation, or under nutrient starvation. In both cases, only soluble proteasomes could serve as a substrate to autophagy, suggesting a modified model whereby substrates for proteaphagy are dysfunctional proteasomes in their near-native soluble state, and not as previously believed, those sequestered at the IPOD. Furthermore, the insoluble fraction accumulating in the IPOD represents an alternative pathway, enabling the removal of inactive proteasomes that escaped proteaphagy when the system became saturated. Altogether, we suggest that the relocalization of proteasomes to soluble aggregates represents a general stage of proteasome recycling through autophagy

Fumarase affects the deoxyribonucleic acid damage response by protecting the mitochondrial desulfurase Nfs1p from modification and inactivation

10.1016/j.isci.2021.103354ISCIENCE241

The Protein Quality Control Machinery Regulates Its Misassembled Proteasome Subunits

<div><p>Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with various aggregation diseases. In eukaryotes, the ubiquitin proteasome system (UPS) plays a vital role in protein quality control (PQC), by selectively targeting misfolded proteins for degradation. While the assembly of the proteasome can be naturally impaired by many factors, the regulatory pathways that mediate the sorting and elimination of misassembled proteasomal subunits are poorly understood. Here, we reveal how the dysfunctional proteasome is controlled by the PQC machinery. We found that among the multilayered quality control mechanisms, UPS mediated degradation of its own misassembled subunits is the favored pathway. We also demonstrated that the Hsp42 chaperone mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments. Thus, we show that proteasome homeostasis is controlled through probing the level of proteasome assembly, and the interplay between UPS mediated degradation or their sorting into distinct cellular compartments.</p></div

(A) Complementation of <i>rpn5ΔC</i> by the wt copy of <i>RPN5</i>. 10-fold serial dilutions of diploid strains that are homozygote (ΔC/ΔC, YSB906), or heterozygote (wt/ΔC, YSB908) for the temperature sensitive allele of <i>RPN5</i> (<i>rpn5</i>Δ<i>C</i>).

<p>A wt diploid (wt/wt, BY4743) was used as a positive control. Cells were spotted on rich medium, and incubated at the semi-permissive (30°C), and restrictive (34°C) temperatures. (B) <b>The wt copy of <i>RPN5</i> in the GFP-<i>RPN5ΔC/RPN5</i>-RFP heterozygous diploid localizes to the nucleus</b>. Logarithmically growing GFP-Rpn5ΔC/Rpn5-RFP heterozygous diploid cells (YSB1056) were grown in galactose containing medium at 30°C. Cells were visualized by DIC, GFP, DAPI and mCherry. (C) <b>Complementation of the temperature sensitive phenotype of <i>rpn5ΔC</i> by the wt copy of <i>RPN5</i> is associated with the elimination of Rpn5ΔC cytosolic aggregates</b>. Similar to B, but using GFP-<i>rpn5ΔC</i>/<i>RPN5</i> heterozygotes diploids (YSB908), and GFP<i>ΔC</i>/GFP-<i>rpn5ΔC</i> homozygous diploids (YSB906) as a control. The graph shows quantitation of the percentage of cells with (blue), or without (w/o) (red) GFP-Rpn5 cytosolic puncta. (D) <b>Degradation of GFP-Rpn5ΔC cytosolic aggregates is mediated by the proteasome</b>. The presence of GFP-Rpn5ΔC signal was visualized in GFP-<i>rpn5ΔC</i>/<i>RPN5</i> heterozygotes diploids (YSB908), in logarithmically growing cells (t-0), and at the indicated time points at 30°C, after the addition of the proteasome inhibitor, MG132, or DMSO (control). The presence or absence of cytosolic puncta was quantitated at the 210 min time point.</p

(A-C) Hsp42 and Hsp26 co-localize with misassembled proteasome lid.

<p>(A,B) Logarithmically growing cells co-expressing GFP<i>-</i>Rpn5ΔC, together with Hsp42-TFP (YSB726) (A), or Hsp26-TFP (YSB903) (B), were grown in galactose containing media at 30°C. (C) Logarithmically growing cells co-expressing rpn5ΔC, <i>Hsp42-TFP and Rpn11-GFP</i> were grown in galactose containing media at 30°C and shifted to 34°C for 3 hrs (YSB577). (D) <b>Co-localization of Hsp42 with the IPOD marker Hsp104 in an <i>rpn5Δc</i> background</b>. Similar to C, but using <i>rpn5ΔC</i> cells co-expressing Hsp42-TFP and Hsp104-GFP (YSB792). (E-F) <b><i>HSP42</i> is essential for the formation of the cytosolic aggregates by misassembled proteasome lid</b>. (E) Similar to A, but in <i>HSP42</i> wt control (YSB1044) (<i>top</i>), and <i>Δhsp42</i> cells (YSB1045) (<i>bottom</i>). The localization of GFP-Rpn5ΔC signal was scored as the percentage of cells showing nuclear localization, or cytosolic puncta (blue and red bars respectively). An mCherry fusion with NIC96, a component of the nuclear pore complex, was used as a nuclear marker. Unless otherwise stated, for each of the graphs, minimum of 200 cells was counted (n >200); error bars show the standard deviation between two independent experiments. (F) Similar to A,B, but in <i>Δhsp42</i> (YSB1002) and <i>HSP42</i> (SB163) control cells grown at 34°C. DAPI was used for nuclear staining. (G) <b>The nuclear relocalization of GFP-Rpn5ΔC in <i>Δhsp42</i> cells is associated with growth restoration</b>. 10-fold serial dilutions of the indicated strains (SB147, SB148, YSB868) were spotted on SC media supplemented with galactose (SC-GAL). Cells were incubated at the semi-permissive (30°C) and restrictive (34°C) temperatures. The wt, and strains harboring <i>rpn5</i>Δ<i>C</i>, were used as positive and negative controls, respectively. (H) <b>The nuclear re-localization of GFP-Rpn5ΔC in Δ<i>hsp42</i> is associated with proteasome reassembly</b>. Similar to 2B, but in cells over producing GFP-<i>Rpn5</i>Δ<i>C</i>, and deleted in the indicated chaperones (YSB219, SB148, YSB868, YSB954, YSB1174 respectively). Cells were grown at the semi-permissive temperature of 30°C, and shifted for 2 hrs to the restrictive temperature (34°C).</p

Models: UPS assembly can be naturally impaired by many factors, and thus, there is competition between assembly, degradation and aggregation of proteasome subunits.

<p>When the proteasome lids are partially misassembled, as demonstrated in <i>rpn5ΔC</i> mutant at the semi-permissive temperature (i), the misassembled subunits are targeted to UPS-mediated degradation by the assembled 26S proteasomes which are still available. This degradation can take place in the nucleus, as recently shown for misfolded proteins [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005178#pgen.1005178.ref044" target="_blank">44</a>]. The employment of <i>rpn5</i>Δ<i>C</i> at the restrictive temperature (ii), shifts the balance to slower assembly, which has a dual effect: It increases the amount of the unassembled subunit, and at the same time decreases its degradation, because there is less proteasomes available. Hence, missassemled proteasome subunits are aggregated in the IPOD, a process that depends mostly on <i>HSP42</i>.</p

(A) The RP subunit Rpn11-RFP co-localizes with GFP-Rpn5ΔC. Logarithmically growing cells containing Rpn11 fused to RFP (Rpn11-RFP) and GFP-Rpn5ΔC (YSB1090) where grown in galactose containing medium at 30°C and shifted to 34°C for 3 hrs.

<p>Cells were visualized by DIC, GFP, and mCherry. (B) <b>Misassembled lid in <i>rpn5</i>Δ<i>C</i> is not associated with the proteasome</b>. Rapidly lysed whole cell extracts from wt (endogenous levels of <i>RPN5</i>) (YSB219), and cells over expressing GFP-<i>RPN5</i>, or GFP-<i>rpn5</i>Δ<i>C</i> (SB147, SB148) thorough a galactose inducible promoter (GFP<i>-RPN5</i>, and GFP-<i>rpn5</i>Δ<i>C</i> respectively) were resolved by nondenaturing PAGE, and proteasome visualized by in-gel peptidase activity. <i>wt</i> proteasomes are found as a mixture of RP2CP and RPCP. Proteasomes in <i>rpn5</i>Δ<i>C</i> mutants migrate faster, pointing to a structural defect. Cells were grown at the semi-permissive (30°C), and samples were then transferred to the restrictive temperature (34°C) for 2 hrs. The GFP-<i>rpn5</i>Δ<i>C/</i>Δ<i>hsp42</i> samples are discussed below. (C,D) <b>Rpn5ΔC cytosolic aggregates co-localize with the IPOD</b>. Logarithmically growing cells co-expressing GFP<i>-</i>Rpn5ΔC, together with the IPOD markers Hsp104-TFP (YSB747) (C) and Rnq1-mCherry (YSB1004) (D), were grown in rich galactose containing medium at 30°C. Hsp104-TFP, and Rnq1-mCherry co-localized with GFP-Rpn5ΔC throughout the experiment. (E) <b>GFP-Rpn5ΔC large cytosolic aggregates remain sequestered to the mother cells</b>. Samples in logarithmic growth at the semi-permissive temperature (30°C) were stained by calcofluor. The localization of GFP-Rpn5ΔC (SB148) signal was scored as the percentage of daughter cells issued from IPOD containing mother cells with (puncta), or without (no puncta) cytosolic puncta. A minimum of 100 cells was counted (n >100); error bars show the standard deviation between two independent experiments.</p