The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-to-ER Retrograde Transport

Abstract

Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human "ubiquitinome" using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport.

Keywords: APP; PSMD14; retrograde; trafficking; ubiquitin.

Figure 1

High-content siRNA screening assay revealed PSMD14 as a novel regulator of amyloid precursor protein (APP) levels. (A) Graphical distribution of the targets evaluated in the primary high content siRNA screening using the siRNA "ubiquitinome" library in H4 cells. (B) Quantification of the total fluorescence intensity of reporter APP-EGFP cells transfected for 72 h with NT siRNA and EGFP siRNA. Bars represent the mean ± SD with a statistical Z factor = 0.69. (C) High content images (20×) captured in reporter APP-EGFP cells transfected for 72 h with non-target (NT) siRNA and EGFP siRNA. (D) Graphical representation of total fluorescence intensity of all 1187 genes analyzed in primary siRNA screening with the reporter APP-EGFP cells. PSMD14 appears indicated as the top hit. (E) High content images (20×) in pseudo color of reporter APP-EGFP cells transfected for 72 h with siRNA SMARTpool targeted against PSMD14 (PSMD14 siRNA) in comparison to cells transfected with NT siRNA. The fluorescence intensity in these images was reduced to avoid saturation with the PSMD14 siRNA. Scale Bar of the images indicates the scale of fluorescence intensity.

Figure 2

PSMD14 is validated as a regulator of the endogenous APP levels. (A) Protein extracts of parental H4 cells either untransfected (Mock), transfected with NT siRNA, or transfected with four different PSMD14 siRNA sequences for 72 h were analyzed by western blot. Polyclonal antibodies to endogenous APP (CT695) and to Ub (that recognizes all types of Ub conjugates), and monoclonal antibodies to PSMD14 (clone D18C7) and to β-actin (clone BA3R), were tested. The position of molecular mass markers are indicated on the left. Densitometric quantification of the levels of endogenous APP (B) and PSMD14 (C) in H4 cells transfected with PSMD14 siRNA#1, compared to untransfected cells (Mock). Statistical significance was determined by Student’s t-test. Bars represent the mean ± SD of biological replicates (APP n =5; PSMD14 n = 4). **p < 0.01 and ***p < 0.001. (D) mRNA levels of psmd14 and (E) mRNA levels of app were measured using RT-qPCR from parental H4 cells transfected for 72 h. All data were normalized for TATA binding protein expression in either untransfected cells (Mock), cells transfected with NT siRNA or cells transfected with four different PSMD14 siRNAs duplexes. Statistical significance was determined by One-Way ANOVA, followed by Tukey’s test. Bars represent the mean ± SD of biological replicates (psmd14 n = 3; app n = 3). Different letters above the mean bars apply to significant differences between groups p < 0.01.

Figure 3

Acute inhibition of PSMD14 by Capzimin CZM shows a similar phenotype as that of PSMD14 KD on the levels of APP and high molecular weight Ub conjugates. (A) Schematic diagram of the molecular targets of Capzimin and MG132 in the 19S RP and 20S catalytic core of the proteasome, respectively. (B) Parental H4 cells were treated either with vehicle (DMSO; Control), or increasing doses of CZM for 4 h, or MG132 for 6 h. Protein extracts were analyzed by western blot with a polyclonal antibody to endogenous APP. Monoclonal antibody to β-actin (clone BA3R) was used as a loading control. The position of molecular mass markers is indicated on the left. (C) Densitometric quantification of APP protein levels as shown in (D). Statistical significance was determined by one-way ANOVA, followed by Tukey’s test. Bars represent the mean ± SD of biological replicates (n = 4). Different letters above the mean bars apply to significant differences between groups p < 0.05. (D) Parental H4 cells were treated as in (B), and the protein extracts were analyzed by western blot with a polyclonal antibody to Ub that recognizes all types of Ub conjugate. Monoclonal antibody to β-actin (clone BA3R) was used as a loading control. The position of molecular mass markers is indicated on the left. (E) Immunofluorescence microscopy images of the cellular localization of Ub in parental H4 cells treated with either the vehicle (DMSO; Control), CZM for 4 h or MG132 for 6 h. Cells were fixed, permeabilized and stained with a mouse monoclonal antibody to Ub (clone P4D1) followed by Alexa-488-conjugated donkey anti-mouse IgG. Scale bar, 10 µm. (n = 3).

Figure 4

Acute inhibition of PSMD14 by CZM triggers the accumulation of APP in a swollen Golgi apparatus. Immunofluorescence analysis of endogenous APP in H4 parental cells treated either with the vehicle (DMSO; Control) (A–C) or CZM (D–F) for 4 h. Cells were fixed, permeabilized, and double stained with a rabbit polyclonal antibody to APP (CT695) (A,D) and a mouse monoclonal antibody to GM130 (clone35/GM130) (B,E), followed by Alexa-594-conjugated donkey anti-Rabbit IgG and Alexa-488-conjugated donkey anti-Mouse IgG. Merging of the images generated the third picture (C,F). Scale bar, 10 mm. (G) Quantitative analysis of the mean of total fluorescence intensity of APP upon treatment with CZM, in comparison to control cells. The statistical significance was determined by Student’s t-test. Bars represent the mean ± SD of the fluorescent signal per cell area (n = 43 cells). ***p < 0.001. (H) Quantitative analysis of the fraction of APP colocalizing with GM130 under CZM treatment and compared to control cells. Statistical significance was determined by Student’s t-test. Bars represent the mean ± SD of the fluorescent signal per cell area (n = 43 cells). ***p < 0.001. (I) Quantitative analysis of the cell area. Statistical significance was determined by Student’s t-test. Bars represent the mean ± SD of the cell area (n = 43 cells) **p < 0.001. (J) Immunofluorescence microscopy analysis of GM130 in parental H4 cells treated either with the vehicle (DMSO; Control) or CZM for 4 h. Cells were fixed, permeabilized and stained with mouse monoclonal antibody to GM130 (clone 35/GM130) followed by Alexa-488-conjugated donkey anti-mouse IgG, and nuclei were stained with DAPI. Scale bar, 10 μm. (K) 3D reconstructions of the Golgi apparatus using GM130 as Golgi marker were generated from Z-stacks (250 nm). (L) Golgi Volume was measured from 3D reconstructions as shown in (K). Statistical significance was determined by Student’s t-test. Bars represent the means ± SEM (n = 20 cells). *** p < 0.001.

Figure 5

The PSMD14 DUB inhibitor CZM impairs Golgi-to-ER retrograde transport. (A) Three-dimensional reconstructions of the Golgi apparatus using giantin as a Golgi marker were generated from Z-stacks (250 nm) obtained from HeLa cells stably expressing KDELR1-GFP treated for 90 min either with vehicle (DMSO; Control), CZM or MG132. (B) Golgi volume was measured from 3D reconstructions as shown in (A). Statistical significance was determined by Student’s t-test. Bars represent the means ± SEM (n = 30 cells). *** p < 0.001. (C) HeLa cells stably expressing KDELR1-GFP were treated for 90 min either with vehicle (DMSO; Control), CZM or MG132. Cells were fixed, and representative confocal images were acquired. (D) Measurement of giantin and total KDELR1-GFP total fluorescent intensity. Statistical significance was determined by Student’s t-test. Bars represent the means ± SEM (n = 34 cells). *** p < 0.001. (E) H4 cells were transiently transfected to express the thermo-sensitive retrograde transport reporter KDELR-VSVG-YFP. Cells were kept at 32 °C to allow KDELR-VSVG-YFP localization at the Golgi. Cells were then shifted to 40 °C (restrictive temperature) and images acquired at 1 min interval for 15 min. (F) Quantitative image analysis was performed to measure the integrated fluorescence of KDELR-VSVG-YFP at the Golgi at 1 min interval for 15 min. Statistical significance was determined by Student’s t-test. Bars represent the mean ± SEM (n = 3 cells). *p < 0.05.

Figure 6

Inhibition of autophagosome formation by CZM. (A) Parental H4 cells were treated either with the vehicle (DMSO; Control), CZM for 4 h or MG132 for 6 h and protein extracts were analyzed by western blot with a polyclonal antibody to LC3B. Monoclonal antibody to β-actin (clone BA3R) was used as a loading control. The positions of the molecular mass markers are indicated on the left. Immunofluorescence microscopy analysis of the subcellular localization of endogenous LC3B in parental H4 cells treated with either the vehicle (DMSO; Control) (B), EBSS for 4 h (C), CZM for 6 h (E) or Torin-1 for 4 h (F). EBSS (D) and Torin-1 (G) were tested using a 2-h pretreatment with CZM followed by the treatment with EBSS or Torin-1 for 4 h in the presence of CZM. Cells were fixed, permeabilized and stained with a rabbit polyclonal antibody to LC3B followed by Alexa-594-conjugated donkey anti-Rabbit IgG, and nuclei were stained with DAPI. Scale bar 10 μm. (H) Quantification of the puncta positive to LC3B. Statistical significance was determined by one-way ANOVA, followed by Tukey’s test. Bars represent the mean ± SEM (n = 50 cells). Different letters above the mean bars indicate the significant differences between groups p < 0.05. (I) Protein extracts from parental H4 cells treated as in (B–G) were analyzed by western blot with a rabbit polyclonal antibody to LC3B. Monoclonal antibody to β-actin (clone BA3R) was used as a loading control. The position of molecular mass markers is indicated on the left. (J) Densitometric quantification of LC3B-I levels and (K) LC3B-II levels. Statistical significance was determined by One-Way ANOVA, followed by Tukey’s test. Bars represent the mean ± SEM of biological replicates (LC3B-I n = 3; LC3B-II n = 3). Different letters above the mean bars indicate the significant differences between groups p < 0.05.

Figure 7

Redistribution of RAB1A and ATG9A to the Golgi apparatus with CZM. (A) Immunofluorescence analysis of endogenous RAB1A and ATG9A in H4 parental cells treated for 4 h either with the vehicle (DMSO; Control) (left panel) or CZM (right panel). Cells were fixed, permeabilized, and stained with a rabbit monoclonal antibody to RAB1A (clone D3X9S) (upper panel) and a rabbit monoclonal antibody to ATG9A (clone EPR2450(2)) (lower panel), followed by Alexa-594-conjugated donkey anti-Rabbit IgG. Scale Bar, 10 µm. (B) Quantitative analysis of the fluorescence intensity of RAB1A upon treatment with CZM, in comparison to control cells. Statistical significance was determined by Student’s t-test. Bars represent the mean ± SEM of the fluorescent signal per cell area (n = 227 cells). **p < 0.01; ***p < 0.001; n.s., not significant. (C) Quantitative analysis of the fluorescence intensity of ATG9A upon treatment with CZM, in comparison to control cells. Statistical significance was determined by Student’s t-test. Bars represent the mean ± SEM of the fluorescent signal per cell area (n = 95 cells). *p < 0.05; ***p < 0.001; n.s., not significant.

Figure 8

Model of the mechanism underlying the regulation of protein membrane trafficking and macroautophagy by the proteasome 19S RP PSMD14 DUB activity. The model depicts the closed interplay between membrane transport and macroautophagy by a novel mechanism involving the proteasome complex through the deubiquitinating activity of PSMD14. We propose that active PSMD14 and the K63-Ub chains (1, left panel) positively regulate Golgi-to-ER retrograde transport (2, left panel), a pathway implicated in the retrieval of key proteins for autophagosome biogenesis and macroautophagy (3, left panel). Reduction of free K63-Ub chains by inactive PSMD14 (1, right panel) results on the blockage of Golgi-to-ER retrograde transport (2, right panel) causing the accumulation of ATG9A and RAB1A at the Golgi apparatus. Thus, blockage of Golgi-to-ER retrograde transport inhibits the biogenesis of autophagosomes and macroautophagy (3, right panel). Macroautophagy has been recently demonstrated to act as a potent positive regulator of protein transport from the Golgi apparatus to the cell surface (Golgi-secretion ON; 4, left panel). Thus, inhibition of macroautophagy upon inactive PSMD14 (siRNA/CZM) blocks protein transport from the Golgi apparatus to the cell surface (Golgi-secretion OFF; 4 right panel), explaining the effect on APP transport (5 left and 5 right panels).