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. 2009 Feb;10(2):173-9.
doi: 10.1038/embor.2008.238. Epub 2009 Jan 16.

PLIC proteins or ubiquilins regulate autophagy-dependent cell survival during nutrient starvation

Elsa-Noah N'Diaye  1 , Kimberly K Kajihara, Ivy Hsieh, Hiroshi Morisaki, Jayanta Debnath, Eric J Brown
Affiliations

PLIC proteins or ubiquilins regulate autophagy-dependent cell survival during nutrient starvation

Elsa-Noah N'Diaye et al. EMBO Rep. 2009 Feb.

Abstract

Ubiquilins (UBQLNs) are adaptor proteins thought to deliver ubiquitinated substrates to proteasomes. Here, we show a role for UBQLN in autophagy: enforced expression of UBQLN protects cells from starvation-induced death, whereas depletion of UBQLN renders cells more susceptible. The UBQLN protective effect requires the autophagy-related genes ATG5 and ATG7, two essential components of autophagy. The ubiquitin-associated domain of UBQLN mediates both its association with autophagosomes and its protective effect against starvation. Depletion of UBQLN delays the delivery of autophagosomes to lysosomes. This study identifies a new role for UBQLN in regulating the maturation of autophagy, expanding the involvement of ubiquitin-related proteins in this process.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Ubiquilins protect against starvation-induced cell death. (A) HeLa cells expressing GFP-tagged UBQLN1 or UBQLN2 (or GFP for controls) were transferred to starvation medium (PBS) for the indicated time. Where specified, only serum was removed from the culture medium. Viability of transfected cells was assessed by PI staining and analysed by flow cytometry. The graph represents the mean of three experiments±s.e. (B) HeLa cells were depleted of UBQLN1 or UBQLN2 by siRNA transfection; depletion was verified by Western blot. Cells were starved for increasing periods of time, and their viability was analysed as in (A). The graph represents the mean of three experiments±s.e; *P<0.05 between control and UBQLN2-KD at 6 h (paired Student's t-test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; KD, knockdown; PI, propidium iodide; siRNA, short interfering RNA; UBQLN, ubiquilin.
Figure 2
Figure 2
The ubiquilin-protective effect against starvation requires autophagy. (A) HeLa cells were transfected with siRNAs for autophagy-related gene ATG5 (siATG5) or with a non-silencing siRNA (siC). Depletion of ATG5, which decreased the levels of the ATG5–ATG12 covalent complex, was confirmed by Western blot. Loading control: transferrin receptor (TfR). (B) Control or ATG5-KD cells transfected with UBQLN1 were starved, and the cell viability was assessed as in Fig 1; the graph represents the mean of three experiments±s.e. (C) HeLa cells were stably transfected with control (shC) or ATG7 (shATG7) shRNA; depletion was verified by Western blot (loading control: GAPDH). (D) Control or ATG7-KD cells transfected with UBQLN1 were starved for 3 or 6 h and assessed for their viability. The graph represents the mean of three experiments±s.e. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KD, knockdown; Pl, propidium iodide; shRNA, short hairpin RNA; siRNA, short interfering RNA; UBQLN, ubiquilin.
Figure 3
Figure 3
Ubiquilins colocalize with autophagosomes. (A) Immunolocalization of transfected Myc-UBQLN (1 or 2) and GFP-tagged microtubule-associated protein 1 light chain 3 (GFP-LC3) was analysed by microscopy. Inset: magnification of UBQLN1 vesicular staining (arrow), which, after acquisition of Z-stacks, was found in all UBQLN structures analysed. (B) Cells were starved for 2 h, with (top row) or without BAF A1 (bottom row). Cells were stained for endogenous UBQLN2 (magenta) and LC3 (green). (C) Control cells (untransfected, right panel) or cells overexpressing Myc-UBQLN2 and GFP-LC3 (left panel) were transferred into starvation medium for 2 h. Localization of Myc-UBQLN2 and GFP-LC3 was analysed by immunoelectron microscopy using Myc (arrow) and GFP (arrowhead) antibodies coupled to 10 and 15 nm gold particles, respectively. Double-membrane vacuoles (autophagosomes) in transfected cells (left panel) show increased staining by both antibodies compared with autophagosomes in untransfected cells (right panel). Scale bar, 0.5 μm. BAF A, bafilomycin A1; GFP, green fluorescent protein; UBQLN, ubiquilin.
Figure 4
Figure 4
The UBA domain of ubiquilin is required for interaction with autophagosomes and a protective effect against starvation. (A) Generation of Myc-tagged UBQLN1 deletion mutants. (B) Each mutant was co-transfected with GFP-tagged microtubule-associated protein 1 light chain 3 (GFP-LC3) and the localization of each protein was analysed by microscopy. (C) Starvation-induced cell death was assessed in cells expressing UBQLN1 or UBQLN1ΔUBA. The graph represents the mean of three experiments±s.e., with *P⩽0.05 (P=0.0017 by one-way ANOVA test, comparing control, UBQLN1 and UBQLN1ΔUBA). No difference was found between control and UBQLN1ΔUBA at any time point. ANOVA, analysis of variance; GFP, green fluorescent protein; Pl, propidium iodide; UBA, ubiquitin-associated; UBQLN, ubiquilin.
Figure 5
Figure 5
Depletion of ubiquilin inhibits the degradation of autophagosomes by lysosomes. (A) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN- or autophagy-related gene ATG5-KD cells expressing GFP-tagged microtubule associated protein 1 light chain 3 (GFP-LC3). (B) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus (in pixels) and by analysing the frequency distribution. (C) Control or UBQLN-KD cells expressing GFP-LC3 were starved and stained with the mitochondria dye Mitotracker™. Dual staining of LC3 (green) with Mitotracker (magenta) was analysed by microscopy. (D) Control and UBQLN-KD cells stained with Mitotracker were starved for 6 h; BAF A was added where indicated. Mitotracker fluorescence was measured by flow cytometry. Unless specified otherwise, values for UBQLN-KD and control+BAF A were compared with that of controls at the same time points, with ***P<0.001, **P<0.01 and *P<0.05 (paired Student's t-test). (E) Cells were transfected with pShooter™ together with control or UBQLN (1+2) siRNA. Expression of pShooter was comparable in control and UBQLN-KD cells (top). Cells were starved for 3 or 6 h, and the intensity of GFP was quantified by flow cytometry (bottom). The fluorescence value obtained with controls (no starvation) was considered as 100%, and the value obtained for the 3- and 6-h time points was normalized to controls. The graph represents the mean±s.e. of four assessments. (F) Cells expressing a chimaeric LC3 fused to both GFP and mCherry were transfected with control or UBQLN siRNA, and starvation was induced for the indicated times. Puncta from 25 cells were scored and analysed by microscopy for their fluorescence colour. The graph represents the mean of three experiments±s.e. The number of mCherry single-positive puncta in UBQLN-KD cells was compared with that of controls for each time point; **P<0.01 and *P<0.05 (paired Student's t-test). (G) Control or UBQLN-KD cells were starved for increasing periods. Where indicated, BAF A was added to the starvation medium (+B). Cell lysates were analysed by Western blot for their content in LC3-II. Loading control: transferrin receptor (TfR). BAF A, bafilomycin A1; GFP, green fluorescent protein; KD, knockdown; siRNA, short interfering RNA; UBQLN, ubiquilin.
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