Page 27 - F. Cell biology

NCLLR regulates non-autophagic LC3 lipidation on the Golgi apparatus through
ATG16L1 recruitment
Jaemin Kang, Cathena Meiling Li, DoHyeong Na, and Yong-Keun Jung
School of Biological Sciences, Seoul National University, Gwanak-ro 1, Gwanak-Gu, Seoul, Korea

BACKGROUND AIM

Autophagy mediates lysosomal degradation of intracellular cargos via autophagy- Here, we identified NCLLR as a novel regulator of LC3 lipidation on the Golgi
related (ATG) proteins. Emerging evidences suggest that ATG proteins also have
autophagy-independent functions in various biological processes. ATG8/LC3 apparatus. This study aimed to investigate the molecular mechanism and the
lipidation not only regulates autophagy but also participates in endocytosis, pathophysiological function of NCLLR-induced LC3 lipidation on the Golgi
phagocytosis and extracellular vesicle secretion. Although recent reports showed apparatus.
that LC3 lipidation occurs on the Golgi apparatus following Golgi damages and
chemical damages, its molecular mechanism and biological function remain largely
unknown.
RESULTS

Figure 1. Overexpression of NCLLR induces LC3 accumulation on Figure 2. LC3 lipidation by ATG5, but not ULK1 complex , is required Figure 3. NCLLR recruits ATG16L1 to the Golgi apparatus through
the trans-Golgi Network for LC3 accumulation by NCLLR interaction with WD40 repeats of ATG16L1

(A, B) Ulk1/2 is not required for LC3 accumulation by NCLLR. Ulk1/2 +/+ and
Ulk1/2 -/- MEFs were transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR)
together with GFP-LC3 for 24 h. Cells were observed under fluorescence
microscope (A) and analyzed by western blotting (B). The percentages of cells
(A, B) LC3 accumulation induced by NCLLR overexpression. HeLa cells were with GFP-LC3 clusters were calculated and presented as mean ± S.D. (*P < 0.05,
transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR) together with GFP-LC3 for * *P < 0.01, ****P < 0.0001, 2way ANOVA followed by Tukey’s multiple
24 h. Cells were observed under fluorescence microscope (A, left) and analyzed by comparisons test, n=3, 100~250 cells) (A, right). (C-F) LC3 I-to-II conversion by (A, B) ATG16L1 is required for LC3 recruitment by NCLLR. HeLa sgCtrl and
sgATG16L1 cells were transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR)
western blotting (B, left). The percentages of cells with GFP-LC3 clusters were Atg5 is required for LC3 recruitment by NCLLR. (C, D) Atg5 WT, KO MEFs were
calculated and presented as mean ± S.D. (****P < 0.0001, 2way ANOVA followed by transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR) together with together together with GFP-LC3 for 24 h. Cells were observed under fluorescence microscope
Tukey’s multiple comparisons test, n=3, 100~200 cells) (A, right). Densitometry with GFP-LC3 for 24 h. Cells were observed under fluorescence microscope (C) (A) and analyzed by western blotting (B). The percentages of cells with GFP-LC3
clusters were calculated and presented as mean ± S.D. (****P < 0.0001, 2way ANOVA
values of (B) were quantified (GFL-LC3 II/I) and presented as mean ± S.D. (*P < 0.05, and analyzed by western blotting (D). The percentages of cells with GFP-LC3
two-tailed t-test, n=3) (B, right). (C,D) LC3 recruitment to the trans-Golgi Network by clusters were calculated and presented as mean ± S.D. (***P < 0.001, 2way followed by Tukey’s multiple comparisons test, n=3, 100~200 cells) (A, right).
NCLLR. (C) HeLa cells were transfected with pcDNA3-HA (Ctrl) or NCLLR-HA ANOVA followed by Tukey’s multiple comparisons test, n=3, 100~250 cells) (C, Densitometry values of (B) were quantified (GFL-LC3 II/I) and presented as mean ±
S.D. (**P < 0.01, ***P < 0.001 two-tailed t-test, n=3) (B, right). (C-E) ATG16L1
(NCLLR) together with TGOLN2-GFP and RFP-LC3 for 24 h. Cells were observed right). (E, F) HeLa cells were transfected with pcDNA3-HA (Ctrl) or NCLLR-HA recruitment to the Golgi apparatus via WD40 repeat by NCLLR. (C, D) HEK293T cells
under confocal microscope. Scale bars represent 10 μm (left). Co-localization of (NCLLR) together with GFP-LC3 WT or G120A for 24 h. Cells were observed
TGOLN2-GFP and RFP-LC3 was quantified by Pearson’s coefficient and presented under fluorescence microscope (E) and analyzed by western blotting (F). The were transfected with indicated cDNA plasmids for 24 h. Cell lysates were subjected to
immunoprecipitation (IP) with FLAG M2 gel (C, left) or anti-HA antibody (C, right, D). (E)
as mean ± S.D. (****P < 0.0001, two-tailed t-test, n=3, 150~250 cells) (right). (D) percentages of cells with GFP-LC3 clusters were calculated and presented as HeLa cells were transfected with indicated cDNA plasmids (Ctrl : pcDNA3-HA, NCLLR :
HeLa cells were transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR) together mean ± S.D. (****P < 0.0001, 2way ANOVA followed by Tukey’s multiple
with GFP-LC3 for 24 h. Immunostaining was performed with anti-GM130 antibody comparisons test, n=3, 100~200 cells) (E, right). Densitometry values of (F) were NCLLR-HA) together with TGOLN2-GFP for 24 h. Cells were observed under confocal
microscope.
and cells were observed under confocal microscope. Scale bars represent 10 μm quantified (GFL-LC3 II/I) and presented as mean ± S.D. (*P < 0.05, two-tailed t-
(left). (E) Endogenous LC3 recruitment to the trans-Golgi Network by NCLLR. HeLa test, n=3) (F, right).
cells were transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR) together with
TGOLN2-GFP for 24 h. Immunostaining was performed with anti-LC3B antibody and Figure 6. Endolysosomal membrane proteins are accumulated on
cells were observed under confocal microscope. Scale bars represent 10 μm (left).
the Golgi apparatus by NCLLR via LC3 lipidation- and V-type
ATPase-independent manner
Figure 5. NCLLR-induced LC3-positive vesicles do not undergo
Figure 4. V-type ATPase regulates LC3 lipidation on the Golgi autophagic degradation through fusion with acidic lysosomes
apparatus by NCLLR
(A-D) V-type ATPase inhibitors inhibit LC3 recruitment by NCLLR. HeLa cells were
transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR) together with GFP-LC3
for 24 h and further incubated with indicated chemicals (DMSO, Bafilomycin A1 (A) LAMP1 localization to the NCLLR-induced LC3 clusters. HeLa cells were
(Baf.A1, 20nM), Chloroquine (CQ, 100uM), Concanamycin A (ConA, 200nM)) for 6 h. transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR) together with LAMP1- (A, B) LC3 lipidation-independent accumulation of endolysosomal membrane proteins by
Cells were observed under fluorescence microscope (A, C) and analyzed by GFP and RFP-LC3 for 24 h and then observed under confocal microscope (left). NCLLR. (A) HeLa cells were transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR)
western blotting (B, D). The percentages of cells with GFP-LC3 clusters were Scale bars represent 10μm. Co-localization of LAMP1-GFP and RFP-LC3 was together with GFP-LC3 for 24 h. Immunostaining was performed with anti-CD63 antibody
calculated and presented as mean ± S.D. (***P < 0.001, ****P < 0.0001, 2way quantified by Pearson’s coefficient and presented as mean ± S.D. (***P < 0.001, and cells were observed under confocal microscope. Scale bars represent 10 μm. (B)
ANOVA followed by Tukey’s multiple comparisons test, n=3, 100~200 cells) (A, C two-tailed t test, n=3, 100~200 cells, right) (B,C) LC3 clusters generated by NCLLR HeLa sgCtrl and sgATG16L1 cells were transfected with pcDNA3-HA (Ctrl) or NCLLR-HA
right). Densitometry values of (B, D) were quantified (GFL-LC3 II/I) and presented fail to fuse with acidic lysosomes. (B) HeLa cells were transfected with pcDNA3-HA (NCLLR) together with RFP-LC3 and TGOLN-GFP (B, left) or GFP-CD63 (B, right) for 24
as mean ± S.D. (n=3) (B, D right) (Ctrl) or NCLLR-HA (NCLLR) together with RFP-LC3 for 24 h and further incubated h. Cells were observed under fluorescence microscope. (C) V-type ATPase-independent
with DQ-Red BSA to stain acidic lysosomes. Cells were observed under confocal accumulation of endolysosomal membrane proteins by NCLLR. HeLa cells were
microscope. Scale bars represent 10μm. (C) HeLa cells were transfected with transfected with pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR) together with RFP-LC3 and
pcDNA3-HA (Ctrl) or NCLLR-HA (NCLLR) together with mCherry-GFP-LC3 for 24 h TGOLN-GFP (C, left) or GFP-CD63 (C, right) for 24 h. After incubation with Bafilomycin
and observed under confocal microscope. Scale bars represent 10 μm. A1 (Baf.A1, 6 h), cells were observed under fluorescence microscope.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
NCLLR overexpression induces LC3 lipidation on the trans- Fletcher, Katherine, et al. "The WD40 domain of ATG16L1 is required for its non‐canonical We would like to thank Noboru mizushima (The University of
Golgi Network via ubiquitin-like protein conjugation system. role in lipidation of LC3 at single membranes." The EMBO journal 37.4 (2018). Tokyo, Japan) for generous gifts of Atg5 WT, KO MEFs.
NCLLR recruits ATG16L1 to the Golgi apparatus for LC3 Galluzzi, Lorenzo, et al. "Molecular definitions of autophagy and related processes." The
lipidation. LC3 vesicles recruited to the Golgi apparatus by EMBO journal 36.13 (2017): 1811-1836.
Gao, Ying, et al. "Golgi-associated LC3 lipidation requires V-ATPase in noncanonical
NCLLR do not undergo lysosomal degradation. V-type autophagy." Cell death & disease 7.8 (2016): e2330.
ATPase regulates the NCLLR-induced LC3 lipidation. Gomes-da-Silva, Lígia C., et al. "Recruitment of LC3 to damaged Golgi apparatus." Cell
NCLLR induced LC3 lipidation- and V-type ATPase- Death & Differentiation 26.8 (2019): 1467-1484.
independent accumulation of endolysosomal membrane Heckmann, Bradlee L., et al. "LC3-Associated Endocytosis Facilitates β-Amyloid Contact information
proteins. These results suggest that NCLLR could be the Clearance and Mitigates Neurodegeneration in Murine Alzheimer’s Disease." Cell (2019).
regulator of non-autophagic LC3 lipidation on the Golgi Levine, Beth, and Guido Kroemer. "Biological functions of autophagy genes: a disease
apparatus. perspective." Cell 176.1-2 (2019): 11-42. Jaemin Kang
Laboratory of Apoptosis & Molecular Disease, School of Biological Sciences, Seoul
Liu, Yajun, et al. "Niclosamide Triggers Non-Canonical LC3 Lipidation." Cells 8.3 (2019):
248. National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Korea
Tel: 82-2-880-4402
E-mail: kjm0831@snu.ac.kr