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. 2011 Feb 1;124(Pt 3):469-82.
doi: 10.1242/jcs.076489.

Autophagic substrate clearance requires activity of the syntaxin-5 SNARE complex

Maurizio Renna  1 , Catherine Schaffner, Ashley R Winslow, Fiona M Menzies, Andrew A Peden, R Andres Floto, David C Rubinsztein
Affiliations

Autophagic substrate clearance requires activity of the syntaxin-5 SNARE complex

Maurizio Renna et al. J Cell Sci. .

Abstract

Autophagy is a lysosome-dependent cellular catabolic mechanism that mediates the turnover of intracellular organelles and long-lived proteins. Reduced autophagic activity has been shown to lead to the accumulation of misfolded proteins in neurons and might be involved in chronic neurodegenerative diseases. Here, we uncover an essential role for the syntaxin-5 SNARE complex in autophagy. Using genetic knockdown, we show that the syntaxin-5 SNARE complex regulates the later stages of autophagy after the initial formation of autophagosomes. This SNARE complex acts on autophagy by regulating ER-to-Golgi transport through the secretory pathway, which is essential for the activity of lysosomal proteases such as cathepsins. Depletion of syntaxin-5 complex components results in the accumulation of autophagosomes as a result of lysosomal dysfunction, leading to decreased degradation of autophagic substrates. Our findings provide a novel link between a fundamental process such as intracellular trafficking and human diseases that might be affected by defective biogenesis and/or homeostasis of the autophagosome-lysosome degradation system.

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Figures

Fig. 1.
Fig. 1.
Knockdown of Sec22B impairs autophagic flux. (A–C) HeLa cells were transfected for 72 hours with 20 nM of control siRNA or siRNA against Sec22B. For the assessment of autophagy by LC3-II levels, a saturating concentration (400 nM) of Bafilomycin A1 (Calbiochem) was added to the cells in the last 4 hours before harvesting. The graphs in B and C report the quantitative analysis of LC3-II and p62 levels relative to actin in three independent experiments performed at least three times in triplicate. The P values for the densitometric analyses were determined by factorial ANOVA test using STATVIEW v4.53 (Abacus Concepts), where the control condition was set to 100. The y-axis values are shown as a percentage and the error bars denote s.e.m. (n=3; ***P<0.001; **P<0.01; NS, non-significant). (D) HeLa cells seeded on glass coverslips were transfected for 96 hours with 20 nM of either anti-Sec22B or control siRNA. In the last 48 hours, cells were retransfected with the same siRNA mix plus 2 μg of the GFP–HD74 expression vector. The P values for assessing aggregation of EGFP–HDQ74 were determined using Student's t-test (n=3; ***P<0.001).
Fig. 2.
Fig. 2.
Knockdown of Sec22B increases autophagosome numbers. (A) HeLa cells were seeded on glass coverslips and transfected for 72 hours with 20 nM of either anti-Sec22B or control siRNA. In the last 24 hours, cells were retransfected with the same siRNA mix plus 0.5 μg of the GFP–LC3 construct. Cells were finally fixed and analysed under fluorescence microscope. The P values for assessing the number of GFP–LC3 dots were determined using Student's t-test (n=3; ***P<0.001). (B,C) HeLa cells stably expressing the mRFP-GFP/LC3 construct were seeded on glass coverslips and transfected for 72 hours with 20 nM of either anti-Sec22B or control siRNA. After 72 hours, cells were fixed and analysed by confocal microscopy. 4× magnifications of the merged channels (insets from A) are shown in B. (D) The P values for assessing the number of LC3-II-positive autolysosomes were determined using Student's t-test (n=3; ***P<0.001). Scale bars: 10 μm.
Fig. 3.
Fig. 3.
Knockdown of Sly1 increases autophagosome numbers. (A) HeLa cells were transfected for 72 hours with 20 nM of either anti-Sly1 or control siRNA. The western blot panels reporting the effect of knockdown of Sly1 on the LC3-II, p62 and Sly1 levels are representative of at least three independent experiments performed in triplicate. (B) HeLa cells were seeded on glass coverslips and transfected for 72 hours with 20 nM of either anti-Sly1 siRNA or control siRNA. In the last 24 hours, cells were retransfected with the same siRNA mixture plus 0.5 μg of the GFP–LC3 construct. Cells were finally fixed and analysed under fluorescence microscope. The P values for assessing the number of GFP–LC3 dots were determined using Student's t-test (n=3; ***P<0.001). (C–E) HeLa cells were transfected for 72 hours with 20 nM of either anti-Sly1 or control siRNA. For the assessment of autophagy by LC3-II levels, a saturating concentration (400 nM) of Bafilomycin A1 (Calbiochem) was added to the cells in the last 4 hours before harvesting. The graphs in D and E show the quantitative analysis of LC3-II and p62 levels relative to actin from independent experiments performed at least three times in triplicate. The P values for the densitometric analyses were determined by factorial ANOVA test using STATVIEW v4.53 (Abacus Concepts), where the control condition was set to 100. The y-axis values are shown in percentage and the error bars denote s.e.m. (n=3; ***P<0.001; *P<0.05; NS, non-significant). (F) HeLa cells seeded on glass coverslips were transfected for 96 hours with 20 nM of either anti-Sly1 siRNA or control siRNA. In the last 48 hours, cells were re-transfected with the same siRNA mix plus 2 μg of the GFP–HD74 expression vector. The P values for assessing EGFP–HDQ74 aggregation were determined using Student's t-test (n=3; **P<0.01).
Fig. 4.
Fig. 4.
Knockdown of Sly1 impairs autophagic flux. (A,B) HeLa cells stably expressing the mRFP-GFP/LC3 construct were seeded on glass coverslips and transfected for 72 hours with 20 nM of either control or anti-Sly1 siRNA. After 72 hours, cells were fixed and analysed by confocal microscope. 4× magnifications of the merged channels (insets from A) are shown in B. (C) The P values for assessing the number of LC3-II-positive autophagosomes and autolysosomes were determined using Student's t-test (n=3; ***P<0.001; **P<0.01). Scale bars: 10 μm.
Fig. 5.
Fig. 5.
Knockdown of syntaxin-5 impairs LC3-II degradation. (A–C,E) HeLa cells were transfected for 72 hours with 20 nM of either control or anti-syntaxin-5 siRNA. For the assessment of autophagy by LC3-II levels, a saturating concentration (400 nM) of Bafilomycin A1 (Calbiochem) was added to the cells in the last 4 hours before harvesting. The graphs in C and E show the quantitative analysis of LC3-II and p62 levels relative to actin in independent experiments performed at least three times in triplicate. The P values for the densitometric analyses were determined by factorial ANOVA test using STATVIEW v4.53 (Abacus Concepts), where the control condition was set to 100. The y-axis values are shown in percentage and the error bars denote s.e.m. (n=3; **P<0.01; NS, non-significant). (D) HeLa cells were seeded on glass coverslips and transfected for 72 hours with 20 nM of either anti-syntaxin-5 siRNA or control siRNA. In the last 24 hours, cells were re-transfected with the same siRNA mix plus 0.5 μg of the GFP–LC3 construct. Cells were finally fixed and analysed under fluorescence microscope. The P values for assessing the number of GFP–LC3 dots were determined using Student's t-test (n=3; ***P<0.001). (F) HeLa cells seeded on glass coverslips were transfected for 96 hours with 20 nM of either anti-syntaxin-5 siRNA or control siRNA. In the last 48 hours, cells were retransfected with the same siRNA mix plus 2 μg of the GFP–HD74 expression vector. The P values for assessing EGFP–HDQ74 aggregation were determined using Student's t-test (n=3; **P<0.01).
Fig. 6.
Fig. 6.
Knockdown of syntaxin-5 impairs autophagic flux. (A,B) HeLa cells stably expressing the mRFP-GFP/LC3 construct were seeded on glass coverslips and transfected for 72 hours with 20 nM of either control or anti-syntaxin-5 siRNA. After 72 hours, cells were fixed and analysed by confocal microscope. 4× magnifications of the merged channels (insets from A) are shown in B. (C) The P values for assessing the number of LC3-II positive autophagosomes and autolysosomes were determined using Student's t-test (n=3; ***P<0.001; **P<0.01). Scale bars: 10 μm.
Fig. 7.
Fig. 7.
Knockdown of syntaxin-5 complex members increases LC3 and LAMP-1 colocalisation. (A,B) HeLa cells stably expressing the mRFP-GFP/LC3 construct were seeded on glass coverslips and transfected for 72 hours with 20 nM of control, anti-Sec22B, anti-Sly1 or anti-syntaxin-5 siRNA. After 72 hours, cells were fixed, made permeable in methanol, stained with an anti-LAMP-1 specific antibody, and finally analysed by confocal microscope. 4× magnifications of the merged channels are shown in B. (C) Quantification of GFP-RFP/LC3 colocalisation with endogenous LAMP-1. The analysis was performed using an ImageJ plug-in (JaCoP). The graph shows the Pearson's correlation (blue bars) and the Mander's colocalisation coefficient (fraction of LC3-positive structures overlapping LAMP-1-positive structures; black bars). The P values for assessing the GFP-RFP/LC3 and endogenous LAMP-1 colocalisation were determined using Student's t-test (n=10; ***P<0.001; **P<0.01). Scale bars: 10 μm.
Fig. 8.
Fig. 8.
Knockdown of syntaxin-5 complex components increases colocalisation of endogenous LC3 with LAMP-1. (A,B) HeLa cells were seeded on glass coverslips and transfected for 72 hours with 20 nM of control, anti-Sec22B, anti-Sly1 or anti-syntaxin-5 siRNA. After 72 hours, cells were fixed, made permeable in methanol, stained with anti-LC3 and anti-LAMP-1 specific antibodies, and finally analysed by confocal microscope. 4× magnifications of the merged channels are shown in panel B. (C) 200 cells per experimental condition were analysed under fluorescence microscope and number of endogenous LC3 dots were scored in a blinded fashion. The P values for assessing the number of cells showing more than 20 LC3 dots/cell were determined using Student's t-test (n=3; ***P<0.001; **P<0.05). (D) Quantification of colocalisation between endogenous LC3-II and LAMP-1. The analysis was performed using the JaCoP ImageJ plugin. The graph reports the Pearson's correlation (light blue bars) and the Mander's co-localisation coefficient (fraction of LC3 positive structures overlapping LAMP-1 positive structures; black bars). The P values for assessing the endogenous LC3-II and LAMP-1 colocalisation were determined using Student's t-test (n=10; ***P<0.001; **P<0.01). Scale bars: 10 μm.
Fig. 9.
Fig. 9.
Knockdown of syntaxin-5 SNARE complex components affects cathepsin maturation. (A–C) HeLa cells were transfected for 72 hours with 20 nM of anti-Sec22B (A), anti-Sly1 (B) or anti-syntaxin-5 (C) siRNA and the effect on cathepsin B processing, relative to cells transfected with control siRNA was assessed by western blot analysis. The graphs report the quantitative analysis of both precursor and mature forms of cathepsin B relative to actin obtained from independent experiments performed three times in triplicate. The P values for the densitometric analyses were determined by factorial ANOVA test using STATVIEW v4.53 (Abacus Concepts), where the control condition was set to 100. The y-axis values are shown in percentage and the error bars denote s.e.m. (n=3; ***P<0.001; **P<0.01; *P<0.05; NS, non-significant).
Fig. 10.
Fig. 10.
Knockdown of syntaxin-18 does not affect autophagy. (A–C) HeLa cells were transfected for 72 hours with 20 nM of siRNA against syntaxin-18 or Sly1, or control siRNA. For the assessment of autophagy by LC3-II levels, a saturating concentration (400 nM) of Bafilomycin A1 was added to the cells in the last 4 hours before harvesting. The graphs in B and C show the quantitative analysis of LC3-II and p62 levels relative to actin in independent experiments performed at least three times in triplicate. The P values for the densitometric analyses were determined by factorial ANOVA test using STATVIEW v4.53 (Abacus Concepts), where the control condition was set to 100. The y-axis values are shown in percentage and the error bars denote s.e.m. (n=3; ***P<0.001; **P<0.01; NS, non-significant). (D) HeLa cells seeded on glass coverslips were transfected for 96 hours with 20 nM of anti-syntaxin-18, anti-Sly1 or control siRNA. In the last 48 hours, cells were retransfected with the same siRNA mix plus 2 μg of the GFP–HD74 expression vector. The P values for assessing EGFP–HDQ74 aggregation were determined using Student's t-test (n=3; **P<0.01; NS, non-significant). (E,F) HeLa cells were transfected for 72 hours with either anti-syntaxin-18, anti-Sly1 or control siRNA. The effect exerted by knockdown of syntaxin-18, Sly1 protein levels (E) and processing of cathepsin B, D and L (F), was assessed by western blot analysis. The blots reported are representative of three independent experiments. p, pro form of cathepsin; m, mature form of cathepsin; i, intermediate form (for cathepsin L).
Fig. 11.
Fig. 11.
Knockdown of Sec22B or Sly1 impairs lysosomal degradative activity. (A,B) HeLa cells were transfected for 96 hours with with 20 nM of siRNA against Sec22B, Sly1, syntaxin-18 or control siRNA. In the last 48 hours, cells were retransfected with the same siRNA mix plus 1 μg of the Fcγ-RI-γ expression vector. To allow phagocytosis of IgG-coated beads, transfected HeLa cells were incubated (1 hour; 37°C) with fluorescent beads (Polysciences) covalently conjugated with human polyclonal IgG and ovalbumin (OVA), thoroughly washed and then incubated for a further 24 hours to permit degradation of internalised beads. Cells were then placed at 4°C to prevent internalisation, stained with an anti-human-IgG antibody (Jackson ImmunoResearch) to mark non-internalised beads and then fixed. Internalised beads were subsequently recovered by cell disruption and then incubated with OVA-specific FITC-conjugated antibody (Abcam). The amount of remaining bead-associated OVA was finally quantified by flow cytometry. As an internal standard, treatment of control siRNA transfected cells with BafA1 significantly reduced OVA degradation (blue bar). The P values for the rate of degradation of OVA-coated beads were determined using Student's t-test on three independent experiments performed in triplicate (n=3; ***P<0.001; **P<0.01; *P<0.05; NS, non-significant).
Fig. 12.
Fig. 12.
Schematic representation of effect of depletion of the syntaxin-5 complex on the autophagic pathway. The endoplasmic reticulum is the site of synthesis and maturation of proteins entering the secretory pathway. Depletion of the syntaxin-5 SNARE complex impairs the ER-to-Golgi transport normally occurring in eukaryotic cells (light blue arrows). As a consequence, the anterograde transport and therefore the activity of lysosomal proteases is reduced, resulting in the lysosomal compartment dysfunction, accumulation of autophagosomes and decreased degradation of autophagic substrates.
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