Autophagy

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/kaup20

Membrane Atg8ylation, stress granule formation,

and MTOR regulation during lysosomal damage

Jingyue Jia, Fulong Wang, Zambarlal Bhujabal, Ryan Peters, Michal Mudd,
Thabata Duque, Lee Allers, Ruheena Javed, Michelle Salemi, Christian
Behrends, Brett Phinney, Terje Johansen & Vojo Deretic

To cite this article: Jingyue Jia, Fulong Wang, Zambarlal Bhujabal, Ryan Peters, Michal
Mudd, Thabata Duque, Lee Allers, Ruheena Javed, Michelle Salemi, Christian Behrends,
Brett Phinney, Terje Johansen & Vojo Deretic (2023) Membrane Atg8ylation, stress granule
formation, and MTOR regulation during lysosomal damage, Autophagy, 19:6, 1893-1895, DOI:
10.1080/15548627.2022.2148900

To link to this article: https://doi.org/10.1080/15548627.2022.2148900

Published online: 29 Nov 2022.

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AUTOPHAGY
2023, VOL. 19, NO. 6, 1893–1895
https://doi.org/10.1080/15548627.2022.2148900

AUTOPHAGIC PUNCTUM

Membrane Atg8ylation, stress granule formation, and MTOR regulation during

lysosomal damage
Jingyue Jiaa,b*, Fulong Wanga,b, Zambarlal Bhujabalc, Ryan Petersa,b, Michal Mudda,b, Thabata Duquea,b, Lee Allersa,b,
Ruheena Javeda,b, Michelle Salemid, Christian Behrendse, Brett Phinneyd, Terje Johansenc, and Vojo Deretic a,b*
a
University of New Mexico Health Sicences Center, Albuquerque, New Mexico, USA; bDepartment of Molecular Genetics and Microbiology,
University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA; cMolecular Cancer Research Group, Institute of Medical Biology,
the Arctic University of Norway, Tromsø, Norway; dProteomics Core Facility, UC Davis Genome Center, University of California, Davis, California, USA;
e
Munich Cluster of Systems Neurology, Ludwig-Maximilians-Universität München, München, Germany

ABSTRACT ARTICLE HISTORY

The functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and Received 7 November 2022
include processes collectively referred to as Atg8ylation. Global modulation of protein synthesis Revised 14 November 2022
under stress conditions is governed by MTOR and liquid-liquid phase separated condensates contain­ Accepted 11 November 2022
ing ribonucleoprotein particles known as stress granules (SGs). We report that lysosomal damage KEYWORDS
induces SGs acting as a hitherto unappreciated inhibitor of protein translation via EIF2A/eIF2α Atg8ylation; integrated
phosphorylation while favoring an ATF4-dependent integrated stress response. SGs are induced by stress response; lysosomal
lysosome-damaging agents, SARS-CoV-2 open reading frame 3a protein (ORF3a) expression, damage; Mycobacterium
Mycobacterium tuberculosis infection, and exposure to proteopathic MAPT/tau. Proteomic studies tuberculosis; MTOR; NUFIP2;
revealed recruitment to damaged lysosomes of the core SG proteins NUFIP2 and G3BP1 along with PKR; proteopathic tau; SARS-
the GABARAPs of the mATG8 family. The recruitment of these proteins is independent of SG CoV-2 ORF3a; stress granules
condensates or canonical autophagy. GABARAPs interact directly with NUFIP2 and G3BP1 whereas
Atg8ylation is needed for their recruitment to damaged lysosomes. At the lysosome, NUFIP2
contributes to MTOR inactivation together with LGALS8 (galectin 8) via the Ragulator-RRAGA-
RRAGB complex. The separable functions of NUFIP2 and G3BP1 in SG formation vis-a-vis their role
in MTOR inactivation are governed by GABARAP and Atg8ylation. Thus, cells employ membrane
Atg8ylation to control and coordinate SG and MTOR responses to lysosomal damage.

Abbreviations: Atg8: autophagy related 8; ATG: autophagy related; ATF4: activating transcription
factor 4; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; GABARAP: GABA type A receptor-
associated protein; G3BP1: G3BP stress granule assembly factor 1; LLOMe: L-leucyl-L-leucine methyl
ester; LysoIP: lysosome immunopurification; mRNA: messenger ribonucleic acid; MTOR: mechanistic
target of rapamycin kinase; NUFIP2: nuclear FMR1 interacting protein 2; ORF3a: open reading frame
3a protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SG: stress granule; TIA1:
TIA1 cytotoxic granule associated RNA binding protein

Lysosomes, membrane-bound degradative organelles, provide cardinal metabolic regulator controlling a number of cellu­
a spectrum of housekeeping activities and serve as critical lar processes including protein synthesis. SGs provide an
signaling hubs. Therefore, the integrity of lysosomes is critical additional level of control of protein synthesis as
for cellular homeostasis. Due to the nature of their function, a component of an integrated stress response. SGs contri­
lysosomes are exposed to many extrinsic and intrinsic dama­ bute to generalized translational arrest upon stress trans­
ging agents. These include microbial pathogens, environmen­ duced by several kinases that converge upon and
tal agents, toxic protein aggregates, endogenous crystals phosphorylate EIF2A. Phosphorylation of EIF2A results in
composed of cholesterol or uric acid, and a variety of lysoso­ reduction of general protein synthesis while favoring
motropic drugs, all inducing lysosomal membrane damage. expression of integrated stress response proteins including
Lysosomal damage has been implicated in many human dis­ transcription factor ATF4. The mRNA sequestered within
eases such as infections, neurodegeneration, autoimmunity, SGs can reenter translation upon removal of environmental
metabolic disorders, and cancer, as well as in normal aging. stressors and cell recovery.
Thus, it is important to delineate homeostatic activities coun­ We found that lysosomal damage induces SG formation
tering lysosomal damage. using the conventional markers of SGs such as G3BP1, TIA1
Cells elicit a complex set of responses to lysosomal and mRNA by high content microscopy in a panel of cell lines
damage such as inactivation of MTOR. MTOR is the and primary cells in response to the treatment with lysosomal

CONTACT Vojo Deretic vderetic@salud.unm.edu Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center,
915 Camino de Salud, NE, Albuquerque, New Mexico 87131, USA

*Corresponding author
© 2022 Informa UK Limited, trading as Taylor & Francis Group
1894 J. JIA ET AL.

Figure 1. Lysosomal damage results in generalized protein synthesis inhibition by induction of stress granule (SG) formation and MTOR inactivation. The signal from
damaged lysosomes is transduced to the SG machinery via a pool of EIF2AK2/PKR that resides on lysosomes. Upon damage, PKR phosphorylates EIF2A/eIF2α, a key
regulator of translational arrest and SG formation. Inhibition of capped mRNA translation due to EIF2A phosphorylation and SG formation elicits an ATF4-driven
integrated stress response in response to lysosomal damage. Complementary to SGs, lysosomal damage also inactivates MTOR. Under normal conditions, MTOR
phosphorylates its substrates RPS6KB1/S6K1 and EIF4EBP1 to promote protein translation. Damaged lysosomes are Atg8ylated by GABARAPs which directly bind and
recruit SG proteins NUFIP2 and G3BP1 to the lysosomal surface where they act to inactivate MTOR. The pools of NUFIP2 and G3BP1 are split between SG formation
and the Ragulator-RRAG complex to control MTOR activity together with LGALS8 on the lysosomes. This results in both a competition (limiting SG formation) and
a synergy (inhibiting translation by both sequestering mRNAs in SGs and inhibiting MTOR) thus coordinating these two roles of NUFIP2 and G3BP1 for the optimal
contribution of both aspects of protein translation.

damaging agents including Leu-Leu-OMe (LLOMe), glycyl- The enhanced levels of NUFIP2, G3BP1 and TIA1 in LysoIP
L-phenylalanine 2-naphthylamide and silica crystals [1] preparations from cells treated with LLOMe are not inhibited by
(Figure 1). The hallmark of a conventional SG response, cycloheximide, confirming that the recruitment of individual SG
EIF2A phosphorylation, is detected in response to lysosomal proteins to damaged lysosomes is independent of SG formation.
damage. Among the known kinases phosphorylating EIF2A, What might be the functions of individual SG proteins
only EIF2AK2/PKR knockdown abrogates EIF2A phosphor­ on the surface of lysosomes? We found that NUFIP2, the
ylation and SG formation during lysosomal damage. The SG essential component of SGs, translocates from the nucleus
response subsides during recovery from lysosomal damage. to lysosomes upon damage, and that it is responsible for
SG formation triggered by lysosomal damage is blocked by MTOR inactivation. We have previously shown that MTOR
cycloheximide, a known inhibitor of SG condensate formation is inactivated by LGALS8 during lysosomal damage via the
in response to standard SG-induced stressors such as arsenite. Ragulator-RRAGA-RRAGB system, and this is reflected in
We found that LLOMe treatment causes general translational our LysoIP proteomic analysis indicating that MTOR dis­
shutdown documented by a puromycin incorporation assay, sociates from damaged lysosomes. Using an established
whereas it selectively increases expression of ATF4. Thus, we assay for measuring the activation state of the Ragulator
conclude that lysosomal damage is a newly identified stimulus guanine nucleotide exchange factor/GEF complex with
for induction of SGs causing generalized translational arrest RRAGs, we found that NUFIP2 together with LGALS8
while promoting an integrated stress response. These relation­ causes RRAGA-RRAGB inactivation thus inhibiting
ships are observed under physiological conditions including MTOR during lysosomal damage.
Mycobacterium tuberculosis infection, SARS-CoV-2 ORF3a Our LysoIP proteomic analysis furthermore revealed
expression, and exposure to a proteotoxic form of MAPT/tau. enrichment of mATG8s (MAP1LC3/LC3s and
We preformed quantitative proteomic analyses of damaged vs. GABARAPs) on damaged lysosomes. We found by GST
undamaged lysosomes using a well-established lysosome immu­ affinity isolation that NUFIP2 interacts directly with
nopurification (LysoIP) protocol. The LysoIP proteomics and GABARAPs but not with LC3s. Further LysoIP proteomic
immunoblotting analyses revealed increased association of indi­ and immunoblotting analyses show that GABARAPs are
vidual SG proteins including G3BP1, TIA1 and NUFIP2 with responsible for the recruitment of NUFIP2 to damaged
damaged lysosomes. We observed by imaging analysis only a low- lysosomes, and that this is key to MTOR inactivation. The
level association between SGs and lysosomes, indicating that conjugation of GABARAPs to damaged lysosomal mem­
individual SG proteins on damaged lysosomes may have addi­ branes as a manifestation of Atg8ylation responses during
tional functions independent of the roles they play within SGs. stress have a balancing effect on MTOR inactivation vs. SG
 AUTOPHAGY 1895

formation. Thus, we found that Atg8ylation controls both Funding

SG formation and MTOR inactivation during lysosomal
This work was supported by the National Institute of General Medical
damage, balancing via a shared factor, NUFIP2, the two Sciences [P20GM121176]; National Institute of Allergy and Infectious
key aspects of translational arrest. Diseases [R37AI042999, R01AI111935].
In conclusion, this study shows that lysosomal damage is
a hitherto unknown inducer of SG formation and integrated stress
response and that membrane Atg8ylation coordinates SG forma­ ORCID
tion with MTOR inactivation in response to lysosomal damage Vojo Deretic http://orcid.org/0000-0002-3624-5208
affecting protein translation regulation, which is of relevance for
multiple physiological conditions and disease states.
Reference

Disclosure statement [1] Jia J, Wang F, Bhujabal Z, et al. Stress granules and mTOR are
regulated by membrane atg8ylation during lysosomal damage.
No potential conflict of interest was reported by the author(s). J Cell Biol. 2022 Sep 30;221(11):e202207091.