Biology subjects

Lystad, A. H.

Publications and source records attributed to Lystad, A. H..

6 recordsLinked to original sources

bioRxiv2026

ATG16L2 creates a VAIL-dedicated heterodimer with ATG16L1

The ATG8 conjugation machinery supports both canonical autophagy, in which ATG8 proteins are lipidated on forming autophagosomes, and CASM, in which ATG8 proteins are conjugated to stressed single-membrane compartments. How cells allocate this shared machinery between these competing membrane programs remains unclear. Here, we identify ATG16L2 as a specialized regulator of V-ATPase-responsive ATG8 lipidation (atg8ylation). ATG16L2 does not form stable homodimers and is inactive in the absence of ATG16L1, but it becomes functional through heterodimerization with ATG16L1. The ATG16L1-ATG16L2 heterodimer constitutes a VAIL-competent E3-like complex that is excluded from WIPI2-positive autophagic membranes but readily recruited to stressed lysosomes. ATG16L2 also confers VAIL activity onto the otherwise VAIL-deficient ATG16L1 isoform. We propose that ATG16L1 homodimers and ATG16L1-ATG16L2 heterodimers represent alternative assemblies of the ATG8 conjugation machinery: ATG16L1 homodimers are dedicated to WIPI2-positive autophagic membranes, whereas ATG16L1-ATG16L2 heterodimers containing either ATG16L1 isoform form a VAIL-selective assembly.

molecular biology↗

bioRxiv2022

Lysosome repair by ER-mediated cholesterol transfer

Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by formation of contacts with the endoplasmic reticulum (ER), which depend on the ER proteins VAPA/B. The cholesterol-binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP-ORP1L interactions. The PtdIns 4-kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage-induced accumulation of ORP1L and cholesterol and led to failure of lysosomal membrane repair. The cholesterol-PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.

cell biology↗

bioRxiv2020

GAK and PRKCD are positive regulators of PRKN-independent mitophagy

The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy in response to different stimuli remains elusive. Here, we have screened for regulators of PRKN-independent mitophagy using an siRNA library targeting 197 proteins containing lipid interacting domains. We identify Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCD) as novel regulators of PRKN-independent mitophagy, with both being dispensable for PRKN-dependent mitophagy and starvation-induced autophagy. We demonstrate that the kinase activity of both GAK and PRKCD are required for efficient mitophagy in vitro, that PRKCD is present on mitochondria, and that PRKCD is required for ULK1/ATG13 recruitment to early autophagic structures. Importantly, we demonstrate in vivo relevance for both kinases in the regulation of basal mitophagy. Knockdown of GAK homologue (gakh-1) in C.elegans or PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy.

cell biology↗

bioRxiv2020

The Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy

Autophagy plays an essential role in the defence against many microbial pathogens as a regulator of both innate and adaptive immunity. Among some pathogens, sophisticated mechanisms have evolved that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy subversion mediated by the recently discovered Vibrio cholerae cytotoxin, MakA. pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex required for LC3 lipidation at the membranous aggregate resulted in an inhibition of both canonical autophagy and autophagy-related processes including the unconventional secretion of IL-1β. These findings identify a novel mechanism of host autophagy subversion and immune modulation employed by V. cholerae during bacterial infection.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

Autophagy is a fundamental catabolic process essential for development, homeostasis and proper immune function 1. During autophagy, a cascade of ATG proteins target intracellular cargoes for lysosomal degradation and recycling 2. This pathway utilises a unique post-translational modification, the conjugation of ATG8 proteins to phosphatidylethanolamine (PE) at autophagosomes, which modulates cargo selection and maturation. ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving Single Membrane ATG8 Conjugation (SMAC) to endolysosomal compartments, which plays a key role in phagocytosis and other processes 3. It has been widely assumed that SMAC involves the same lipidation of ATG8 to PE, but this has yet to be formally tested. Here, we show that ATG8 undergoes alternative lipidation to phosphatidylserine (PS) during non-canonical autophagy/SMAC. Using mass spectrometry, we find that activation of SMAC, by pharmacological agents 4,5, or during non-canonical autophagy processes such as LC3-associated phagocytosis 6,7 and Influenza A virus infection 8, induces the covalent conjugation of ATG8 to PS, as well as PE. This alternative lipidation event is dependent on the ATG16L1 WD40 domain, and occurs at PS enriched endolysosomal membranes. Importantly, we find that the ATG8-PS and ATG8-PE adducts are differentially delipidated by isoforms of the ATG4 family, indicating significant molecular distinctions and mechanisms between these two species. Together, these results provide an important new insight into autophagy signalling, revealing an alternative form of the hallmark ATG8-lipidation event, so widely used to define and assay autophagy. Furthermore, ATG8-PS lipidation provides a specific molecular signature for non-canonical autophagy, uncovering a novel means of detecting and monitoring this emerging pathway.

cell biology↗

bioRxiv2019

TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls autophagosome shedding by ATG4 protease

Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surface-exposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autoph-agosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation.

biochemistry↗