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. 2023 Sep 11;26(10):107886.
doi: 10.1016/j.isci.2023.107886. eCollection 2023 Oct 20.

AKIR-1 regulates proteasome subcellular function in Caenorhabditis elegans

Johanna Pispa  1 , Elisa Mikkonen  1 , Leena Arpalahti  1 , Congyu Jin  2 , Carmen Martínez-Fernández  3 , Julián Cerón  3 , Carina I Holmberg  1
Affiliations

AKIR-1 regulates proteasome subcellular function in Caenorhabditis elegans

Johanna Pispa et al. iScience. .

Abstract

Polyubiquitinated proteins are primarily degraded by the ubiquitin-proteasome system (UPS). Proteasomes are present both in the cytoplasm and nucleus. Here, we investigated mechanisms coordinating proteasome subcellular localization and activity in a multicellular organism. We identified the nuclear protein-encoding gene akir-1 as a proteasome regulator in a genome-wide Caenorhabditis elegans RNAi screen. We demonstrate that depletion of akir-1 causes nuclear accumulation of endogenous polyubiquitinated proteins in intestinal cells, concomitant with slower in vivo proteasomal degradation in this subcellular compartment. Remarkably, akir-1 is essential for nuclear localization of proteasomes both in oocytes and intestinal cells but affects differentially the subcellular distribution of polyubiquitinated proteins. We further reveal that importin ima-3 genetically interacts with akir-1 and influences nuclear localization of a polyubiquitin-binding reporter. Our study shows that the conserved AKIR-1 is an important regulator of the subcellular function of proteasomes in a multicellular organism, suggesting a role for AKIR-1 in proteostasis maintenance.

Keywords: Biochemistry; Cell biology; Genomics.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Loss of akir-1 results in nuclear accumulation of polyubiquitinated proteins in C. elegans intestinal cells (A) Representative fluorescence micrographs of control and akir-1 RNAi-treated N2 animals expressing the polyubiquitin (polyUb) reporter (vha-6p::UIM2-ZsProSensor) in the intestinal cells (left panels). Merge represents overlay of fluorescence and bright-field images (right panels). Scale bar, 200 μm. (B) Representative confocal micrographs of control and akir-1 RNAi-treated polyubiquitin reporter animals with Hoechst-visualized nuclei. Insets show enlargements. Scale bar, 20 μm. (C and D) Representative confocal micrographs of polyubiquitin immunostaining (polyUb Ab) in dissected intestines of control and akir-1 RNAi-treated wild-type (N2) animals (C), and in control (N2) animals and akir-1(gk528) mutants (D). Nuclei are visualized with Hoechst. Scale bars, 10 μm. The graphs (on right) show visually quantified nuclear polyubiquitin accumulation in the intestine in control and akir-1 RNAi-treated wild-type animals (C), and in control (N2) and akir-1(gk528) animals (D). n = total number of animals. Proportions of animals with strongly positive, weakly positive, or negative (i.e., less or equal to cytoplasmic immunostaining) nuclear immunostaining are indicated in percentages. See also Figure S1.
Figure 2
Figure 2
Knockdown of akir-1 alters proteasome activity (A and B) Western blot analysis with antibody against proteasomal 20S alpha subunits using lysates of akir-1(gk528) mutants and control (N2) animals (A), and of control and akir-1 RNAi-treated wild-type animals (B). Anti-alpha-tubulin antibody was used as a normalization control. The quantification graphs show the average fold change in akir-1(gk528) mutants compared to control (N2) animals (set to 1; n = 6 independent experiments) (A), and in akir-1 RNAi-treated wild-type animals compared to control RNAi-treated animals (set to 1; n = 8 independent experiments) (B). (C) In-gel proteasome activity assay with whole animal lysates of wild-type animals exposed to control or akir-1 RNAi treatment (upper gel). Coomassie staining of the same gel (lower gel). The quantifications show the average fold change in chymotrypsin activity in akir-1 RNAi-treated wild-type animals compared to control RNAi-treated animals (set to 1; n = 5 independent experiments). Proteasome activity is indicated as total (Total; CP + RP-CP + RP2-CP), as core particle (CP), and as CP activity subtracted from total activity (Total − CP). RP, Regulatory particle. (D) Immunoblot analysis of whole animal lysates separated under non-denaturing condition using the antibody against proteasomal 20S alpha subunits. Ponceau S staining was used for total protein normalization. The quantification graph shows the average fold change of core particles (CP) in akir-1 RNAi-treated wild-type animals compared to control RNAi-treated animals (set to 1; n = 3 independent experiments). (E) Representative fluorescence micrographs of control and akir-1 RNAi-treated transgenic C. elegans expressing photoconvertible UbG76V-Dendra2 reporter (vha-6p::UbG76V::Dendra2) in intestinal cells. The 0 h (left panels) and 18 h (right panels) indicate time after photoconversion. Scale bar, 500 μm. The graph shows the mean percentages of fluorescence intensity of the photoconverted UbG76V-Dendra2 18 h after the photoconversion relative to the fluorescence at the point of photoconversion (0 h, set as 100%); n = 6 independent experiments with triplicate images of 6–7 animals per image (total number of animals is 108 per treatment). (F) Representative confocal fluorescence micrographs of intestinal cells with photoconverted UbG76V-Dendra2 (18 h after conversion) in transgenic C. elegans treated with control or akir-1 RNAi. Scale bar, 10 μm. The graph shows the ratio between nuclear and cytoplasmic mean fluorescence per cell. n = 2 independent experiments (total number of nuclei is 23 in control RNAi and 27 in akir-1 RNAi treatment). Welch’s t-test (two-tailed distribution and unequal variance) was used for statistical analyses. Error bars, SD; ns, not significant; ∗p < 0,05; ∗∗p < 0,01; ∗∗∗p < 0.001. See also Figure S2.
Figure 3
Figure 3
Intestinal nuclei display reduced proteasome levels upon akir-1 depletion (A) Representative micrographs of proteasome immunostaining (20S Ab) in dissected intestines of control (N2) animals and akir-1(gk528) mutants. The graph shows the normalized mean ± SD intensity profiles of 20S immunofluorescence measured along the line intersecting the cytoplasm and the nucleus as shown in the image above the graph. Orange line represents the profiling line, dashed line represents the nucleus. During the profiling, Hoechst signal was used to determine the nuclear 20S immunofluorescence. n = total number of nuclei is 26 in control (N2) and 44 in akir-1(gk528) animals. (B) Representative confocal micrographs showing GFP fluorescence ratio between nuclei and cytoplasm of control and akir-1 RNAi-treated rpt-5p::GFP::RPT-5 animals. Intestinal cells are outlined with white dashed lines and white arrows point to intestinal cell nuclei (C) Representative confocal micrographs of ubh-4p::UBH-4::GFP animals. Nuclei are visualized with Hoechst. The graphs show the normalized mean ± SD intensity profiles of fluorescence measured along the line intersecting the cytoplasm and the nucleus. n = total number of nuclei is 13 in control RNAi and 17 in akir-1 RNAi treatment (B) and 8 nuclei in control RNAi and 9 nuclei in akir-1 RNAi treatment (C). Scale bars, 20 μm. Error bars, SD. See also Figure S3.
Figure 4
Figure 4
Nuclear proteasome expression decreases in oocytes upon akir-1 depletion (A) Representative confocal micrographs of proteasome immunostaining (20S Ab) in dissected oocytes of control (N2) animals and akir-1(gk528) mutants. The graphs show the normalized mean ± SD intensity profiles of 20S immunofluorescence measured along the line intersecting the cytoplasm and the nucleus. n = total number of nuclei is 10 in control (N2) and 11 in akir-1(gk528) animals. (B) Representative confocal micrographs showing GFP fluorescence in oocytes of control and akir-1 RNAi-treated ubh-4p::UBH-4::GFP animals. The graph shows the normalized mean ± SD intensity profiles of fluorescence measured along the line intersecting the cytoplasm and the nucleus. n = total number of nuclei is 14 in control RNAi and 10 in akir-1 RNAi treatment. White arrows point to oocyte nuclei. Nuclei are visualized with Hoechst. Scale bars, 10 μm. Error bars, SD. See also Figure S4.
Figure 5
Figure 5
The effects of akir-1 depletion on nuclear accumulation of polyubiquitinated proteins in oocytes and body-wall muscle cells (A) Representative micrographs of polyubiquitin immunostaining (polyUb Ab) in dissected oocytes of control (N2) animals and akir-1(gk528) mutants. Nuclei are visualized with Hoechst. Scale bar, 20 μm. White arrows point to representative nuclei. (B) Representative fluorescence micrographs of F1 generation of control and akir-1 RNAi-treated rrf-3(pk1426) animals expressing the polyubiquitin (polyUb) reporter (unc-54p::UIM2-ZsProSensor) in the body-wall muscle cells. Nuclei are visualized with Hoechst. Insets show enlargements of the indicated areas. White arrows point to representative nuclei. Scale bar, 10 μm. The graph (on right) shows quantified subcellular localization of polyUb reporter fluorescence in the body-wall muscle cells. Welch’s t-test (two-tailed distribution and unequal variance) was used for statistical analysis. n = total number of nuclei; ∗∗p < 0,01. See also Figure S5.
Figure 6
Figure 6
Downregulation of akir-1 reduces progeny number and lifespan, and promotes nuclear accumulation of polyubiquitin-binding reporter in intestinal cells even during adulthood (A) The graph shows the mean number of progeny of rrf-3(pk1426) animals exposed to control or akir-1 RNAi treatment. n = 3 independent experiments (total number of P0 animals is 23 for control RNAi and 22 for akir-1 RNAi). Welch’s t-test (two-tailed distribution and unequal variance) was used for statistical analysis. Error bars, SD; ∗∗∗p < 0.001. (B) A representative Kaplan-Meier survival curve from one experiment with rrf-3(pk1426) animals exposed to control or akir-1 RNAi treatment started at the L1 larval stage. (C) The table shows the statistics of three independent lifespan experiments of rrf-3(pk1426) animals treated with control or akir-1 RNAi started at the L1 larval stage, and significance of akir-1 RNAi treatment compared to control RNAi treatment determined with a Mantel-Cox (log rank) test. Mean, restricted mean survival; C.I, confidence interval. (D) Representative fluorescence micrographs of 3-day adult animals expressing the polyubiquitin (polyUb) reporter in intestinal cells (vha-6p::UIM2-ZsProSensor) and treated with control or akir-1 RNAi (left panels) started at the L4 larval stage. Merge represents overlay of fluorescence and bright-field images (right panels). Scale bar, 500 μm. Insets on left panels show representative confocal micrographs of polyubiquitin reporter animals with Hoechst-visualized nuclei. Fluorescence signal in the insets micrographs have been modified differently to show the most representative fluorescence pattern in both treatments. Scale bar, 10 μm.
Figure 7
Figure 7
Distinctly perturbed nuclear transport mimics the akir-1 RNAi-induced polyubiquitin phenotype, but akir-1 is not required for general nuclear import (A) The table shows C. elegans karyopherins, their human orthologues, and RNAi-induced nuclear accumulation of the intestinal polyubiquitin (polyUb) reporter. (B) Representative fluorescence micrographs of control and ima-3 RNAi-treated N2 animals expressing the polyubiquitin (polyUb) reporter (vha-6p::UIM2-ZsProSensor) in the intestinal cells (left panels). Merge represents overlay of fluorescence and bright-field images (right panels). Scale bar, 200 μm. (C) Representative confocal micrographs of control and ima-3 RNAi-treated polyubiquitin reporter animals with Hoechst-visualized nuclei. White arrows point to representative intestinal cell nuclei. Scale bar, 10 μm. (D) Representative bright-field images of control (N2) and akir-1(gk528) mutant animals treated with control or ima-3 RNAi, respectively. Scale bar, 200 μm. (E) Representative fluorescence micrographs of control, akir-1, and ima-3 RNAi-treated wild-type animals expressing ubiquitiously nuclear-localized GFP (sur-5p::NLS-GFP) (left panels). Merge represents overlay of fluorescence and bright-field images (right panels). Lowest panels show enhanced fluorescence signal. Scale bar, 50 μm. See also Figure S7.
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