The unique aspects of Atg5 (Fig. 1A), outside of its role in canonical autophagy, have strong ex vivo and important in vivo phenotypes ⁵². Inactivation of Atg5 in myeloid lineage renders mice excessively susceptible to acute infection with Mycobacterium tuberculosis attributed to excessive inflammation ^{35,46,53–56}, which did not extend to other phases of infection as tested here in a murine model of latent tuberculosis ^77–79 (Fig. S1A-D). In a previous study, we could only partially explain the cellular parameters associated with acute infection, which included degranulation in neutrophils from Atg5^fl/fl LysM-Cre⁺ mice ³⁵. Furthermore, markers of LyHYP (lysosome hypersensitivity phenotype) ³⁵ in ATG5 knockout (KO) cells, such as galectin 3 (Gal3) response, could not be explained by the exocytic phenomena ³⁵. We thus sought to uncover additional processes (Fig. 1A) affected by ATG5 at the intracellular level. To include factors beyond the known processes, we analyzed proteomic data obtained with APEX2-ATG5 ³⁵ and compared APEX2-ATG5^WT with APEX2-ATG5^K130R (an ATG5 mutant deficient in conjugation to ATG12) in cells treated with LLOMe, an agent routinely used to cause lysosomal damage ^80–85 (Figs. 1B and S2A,B). The proximity biotinylation (APEX2)-based proteomic data indicated the presence in the vicinity of APEX2-ATG5 of several protein complexes that control important membrane trafficking pathways including retromer (Fig. 1B). Beside the retromer VPS subunits, there were very few other VPS proteins (Fig. S2C). VPS proteins were observed in ATG5 proteomic data by others ⁸⁶ (Fig. S2D). VPS26 and VPS29 showed statistically significant increase in proximity to ATG5 in cells subjected to LLOMe treatment (Fig. S2A-C). Using APEX2-SGALS1 proximity biotinylation LC-MS/MS as a control, we did not observe enrichment of retromer subunits in cells treated with LLOMe (Fig. S2E).

Association of endogenous ATG5 with endogenous retromer subunits (VPS26A, VPS29 and VPS35) was tested in coimmunoprecipitation (co-IP) experiments (Fig. 1C-E). The interaction between retromer and ATG5 was detected in these experiments and showed increased association in cells subjected to lysosomal damage with LLOMe (Fig. 1C-E). Thus, ATG5, a key component of the known atg8ylation E3 ligases ^42,87, is found in protein complexes with the retromer and this association is enhanced upon lysosomal damage.

During lysosomal damage a number of processes are set in motion to repair, remove and regenerate/replenish lysosomes ^{37,80,88–91} including membrane atg8ylation-dependent processes of repair by ESCRT machinery ⁹² and by lipid transfer via ATG2 ^85,93. We have reported that ATG5 KO cells display elevated Gal3 response to lysosomal damage agents including LLOMe ³⁵. Gal3 is one of the well-characterized sentinel galectins alerting cellular homeostatic systems to lysosomal damage ^{37,80,81,88,94}. Based on the observed interactions between ATG5 and retromer, we tested whether retromer, like ATG5 ³⁵, affected Gal3 response to lysosomal damage. We used the previously established quantitative high content microscopy (HCM) approach, which provides unbiased machine-driven image acquisition and data analysis ^{8,36,80,88,94}. In the experiments herein HCM was based on >500 valid primary objects/cells per well, with a minimum of 5 wells per sample (the independent biological replicates being n≥3) as described ³⁵. Upon treatment with LLOMe, HeLa^VPS35-KO cells had increased Gal3 puncta relative to HeLa^WT cells, comparable to HeLa^ATG5-KO (Fig. 2A,B) ³⁵. The Gal3 phenotype in VPS35 knockout cells was complemented by expression of GFP-VPS35 (Fig. 2C,D). The observed elevated Gal3 recruitment to damaged lysosomes in the absence of VPS35 cannot be explained by increased pools of LAMP2 organelles in the cells, since the overall complement of lysosomes (quantified by LAMP2 puncta/cell) did not increase in LLOMe treated HeLa cells (Fig. S3A,B). Another marker of lysosomal damage, ubiquitination response ⁹⁵, was elevated in both VPS35 and ATG5 deficient cells (Figs. 2E,F). This phenotype was confirmed in another cell line, Huh7 (Fig. S3C,D). Thus, VPS35 and ATG5 defects have a similar effect on lysosomes, previously characterized and termed lysosomal hypersensitivity phenotype/LyHYP ³⁵.

Not all aspects of LyHYP, previously observed in ATG5 knockouts ³⁵, were seen in VPS35 KO cells. In the case of ATG5 inactivation, the recruitment of ALIX, an ESCRT component involved in early stages of lysosomal repair ^80,96,97 is abrogated ³⁵. In ATG5 KO cells (Huh7^ATG5-KO), recruitment of ALIX to lysosomes was prevented, whereas in VPS35 KO cells (Huh7^VPS35-KO and HeLa^VPS35-KO) ALIX was efficiently recruited to lysosomes upon LLOMe treatment (Figs. 2G,H and S3C,D).

The elevated Gal3 and ubiquitin markers of increased lysosomal damage in cells with inactivated VPS35 or ATG5 suggest that both may play a related role in lysosomal resilience to membrane damage, which complements the previously observed effects of aberrant VPS35/retromer on lysosomal morphology and proteolytic capacity ^98,99. The dissimilarities between ATG5 and retromer effects on the ALIX recruitment component can be explained by the previously described perturbed function of the ATG conjugation machinery specifically in ATG5 KO cells affirming the unique additional roles of ATG5 ³⁵. Nevertheless, the striking similarities between ATG5 KO and VPS35 KO effects on heightened Gal3 and ubiquitin responses to lysosomal membrane damage suggest that the two systems (membrane atg8ylation and retromer) closely intersect.

To avoid the confounding, and possibly indirect, effects of lysosomal damage on endolysosomal sorting and trafficking in cells treated with LLOMe, it was necessary to test the effects of ATG5 KO on retromer function under basal conditions with unperturbed lysosomes. Our co-IP analyses (Fig. 1C-E) indicated that ATG5 can be found in protein complexes with retromer subunits even without the lysosomal damage. We further confirmed this in co-IPs showing that GFP-VPS35 and YFP-VPS29 were found in complexes with endogenous ATG5 in resting cells, whereas an isotype IgG control did not pulldown retromer components (Fig. 3A). Reverse co-IPs with GFP-VPS35 and YFP-VPS29 detected endogenous ATG5 and ATG12-ATG5 complexes in GFP/YFP immunoprecipitates (Fig. 3B).

Having established that ATG5 and retromer interact under basal conditions, we tested the effects of ATG5 KO on localization of a well-established cargo for retromer-dependent sorting, GLUT1 ^73,100. We used Huh7 cells, where we generated a panel of membrane atg8ylation and VPS35 CRISPR knockouts (Fig. S4A), because they have a well-defined localization of GLUT1 at the plasma membrane or as intracellular profiles (Fig. 3C), fully amenable (and comparatively more reliable than in HeLa cells) to unbiased quantification by HCM. ATG5 KO in Huh7 cells resulted in redistribution of GLUT1 from the plasma membrane to intracellular punctate profiles (Figs. 3D and S4B). This phenotype was identical to the one observed in VPS35 KO (Fig. 3D and S4B). The increased intracellular GLUT1 puncta overlapped with the lysosomal marker LAMP2 in both ATG5 KO and VPS35 KO cells (Figs. 3C,E and S4C). A similar albeit morphologically less distinct phenotype was observed in HeLa cells (Figs. S4D-H). GLUT1 is sorted by retromer via VPS26’s interactor SNX27, with GLUT1 (and similar cargo) being captured by the PDZ domain of SNX27 ^72,73. In WT cells, SNX27 was found in abundant intracellular profiles (Fig. 3F). In both ATG5 KO and VPS35 KO cells, GLUT1 and SNX27 relocalized to the same intracellular compartment (Fig. 3F,G). We carried out lysosomal purification using the well-established LysoIP method ^80,91,94,101, and quantified levels of GLUT1 and SNX27 in lysosomal preparations that were positive for LAMP2 and devoid of GM130 (Golgi) and PDI (ER) (Fig. 3H). Both GLUT1 and SNX27 were enriched in lysosomal preparations from ATG5 KO and VPS35 KO cells relative to the WT cells (Fig. 3H). These experiments biochemically identify the lysosomes as a compartment to which GLUT1 and SNX27 relocalize in ATG5 KO and VSP35 KO cells. The above findings show that a loss of ATG5 affects retromer-controlled trafficking events.

ATG5 is a part of the membrane atg8ylation machinery ^25,42,87, also known under the term ‘LC3 lipidation’ ¹⁰², which functions within the canonical autophagy pathway but is also engaged in a wide array of noncanonical processes ^{25,38,39,44,103,104}. We tested whether other known components of the atg8ylation apparatus affect retromer. The KOs in Huh7 cells of the components of the prototypical atg8ylation E3 ligase ^25,42 included: ATG5 and ATG16L1, as well as the previously characterized Huh7 lines knocked out for E1 and E2 enzymes, ATG7 ³⁵ and ATG3 ³⁷. These KOs all caused entrapment of GLUT1 in intracellular punctate profiles (Figs. 4A, B) as well as an increase in GLUT1 colocalization with lysosomes (GLUT1⁺ LAMP2⁺ profiles; Fig. 4C,D). We tested whether retromer component levels were altered in atg8ylation mutants and found no change in total cellular levels of VPS26, VPS29 and VPS35, tested in ATG3, ATG5 and ATG7 KO Huh7 cells (Fig. S5A,B) as well as in ATG5 KO HeLa cells (Fig. S5C,D).

Furthermore, comparison of the previously characterized HeLa^Hexa-KO cells ¹⁰⁵, with all principal mATG8s inactivated, with their isogenic parental HeLa^WT cells, showed increased GLUT1 on lysosomes in HeLa^Hexa-KO (GLUT1⁺ LAMP2⁺ profiles; Fig. 4E,F). We observed a similar phenotype in separate triple knockouts (TKOs) of LC3 subfamily and GABARAP subfamily of mATG8s ¹⁰⁵ compared to HeLa^Hexa-KO (Fig. S5B). Thus, the entire membrane atg8ylation machinery was engaged in and required for proper GLUT-1 sorting.

Membrane atg8ylation and mATGs play roles in diverse processes ²⁵ including canonical autophagy ¹. We tested whether the participation of atg8ylation in canonical autophagy, previously reported to affect retromer-dependent cargo sorting under nutrient-limiting conditions ^106,107, is the reason for reduced trafficking of GLUT1 in our experiments. We used KOs in Huh7 cells of ATG13 and FIP200/RB1CC1, two obligatory components of the canonical autophagy initiation machinery ¹, to test whether canonical autophagy under the basal conditions used in our study was responsible for the effects on GLUT1 sorting. We found that in the Huh7 cells knocked out for ATG13 ³⁵ or FIP200 ³⁷ there was no increase in intracellular GLUT1 puncta in contrast to the increase observed with the isogenic ATG5 and VPS35 mutants (Figs. 5A,B). This was mirrored by no increase of GLUT1⁺LAMP2⁺ profiles in ATG13 and FIP200 KO cells whereas colocalization between GLUT1 and LAMP2A increased in ATG5 and VPS35 mutants (Fig. 5BC). To further confirm that autophagy initiation mutant cells retained the ability to perturb GLUT1 trafficking due to defective atg8ylation, we knocked down ATG5 in FIP200 KO cells (Fig. S5D) and found that GLUT1 puncta and GLUT1⁺LAMP2⁺ profiles increased even in the FIP200 KO background to an extend nearing in strength the effects of VPS35 knockout (Figs. 5D-F and S5C). Thus, we conclude that atg8ylation but not canonical autophagy affects retromer function under basal conditions and that a functional atg8ylation apparatus is required for proper sorting of the retromer cargo GLUT1.

Rab7 is considered to be an interactor of the retromer ¹⁰⁸. Rab7 translocates in cells lacking the retromer component VPS35 to lysosomes ¹⁰⁹ including endolysosomal domains with lysosomaly positioned mTORC1 regulatory machinery ¹¹⁰. We thus tested whether absence of ATG5 and components of the atg8ylation apparatus affected Rab7 localization. ATG5, VPS35 as well as knockouts of all major components of the atg8ylation in Huh7 cells, displayed increased Rab7⁺ cytoplasmic profiles (Fig. 6A,B) and overlap between Rab7 and lysosomes (LAMP2) (Figs. 6C and S6A). This was confirmed biochemically using lysosomal purification by LysoIP and immunoblotting for Rab7: there was an increase of Rab7 in lysosomal preparations from ATG5 and VPS35 KO cells (Fig. 6D,E).

This effect extended to other components of the atg8ylation machinery, as KOs in ATG3, ATG7 and ATG16L1 caused a similar effect (Figs. 6A-C and S6A). In contrast, KOs in genes specific for canonical autophagy (ATG13 and FIP200) did not alter intracellular distribution of Rab7 (Figs. 6F-H and S6B). Rab7 distribution was affected by ATG5 knockdown (Fig. S5D) in FIP200 KO cells (Figs. S6B-E). These experiments mirror the effects of ATG5 and atg8ylation on GLUT1 trafficking, showing that ATG5 and atg8ylation machinery but not canonical autophagy is required for proper localization of Rab7.

Membrane atg8ylation has multiple presentations that include not only canonical autophagy but also encompass processes such as CASM ¹⁰³. We thus asked whether induction of CASM ⁹³ could affect GLUT1 trafficking. Using LLOMe as one of the inducers of CASM ⁹³, we detect increased membrane atg8ylation (LC3 puncta formation) in response to lysosomal damage (Fig. 7A,B). When cells were treated with LLOMe ⁹³ and another CASM inducer, monensin ^29,111,112, GLUT1 trafficking to the plasma membrane was negatively affected and instead GLUT1 accumulated intracellularly and trafficked to lysosomes (Figs. 7C-E and S7A). Thus, lysosomal perturbation and induction of CASM may affect retromer-dependent sorting of GLUT1.

It was previously reported that under glucose starvation conditions, LC3A recruits TBC1D5, a GAP for Rab7 ^113–115, away from this small GTPase ¹⁰⁶ and a similar phenomenon could be occurring during CASM. However, under CASM-inducing conditions, no change in interactions between TBC1D5 and LC3A were detected (Fig. S7B,C) suggesting that CASM does not affect the status of the TBC1D5-Rab7 system. Moreover, expression of constitutively active Rab7^Q67L did not promote trafficking of GLUT1 to plasma membrane but rather caused accumulation of intracellular GLUT1 puncta (Fig. 7F-H).

A complication of interpreting CASM as a membrane atg8ylation process responsible for effects on retromer-dependent sorting is that CASM is elicited by lysosomal perturbations using lysosomal pH modifying agents ^{29,103,111,112}, and lysosomal stress could be an independent factor preventing proper sorting of GLUT1. Endolysosomal membrane damage (LLOMe) or perturbations of luminal pH (monensin, bafilomycin A1) negatively affected GLUT1 sorting and caused it to accumulate in lysosomes (Fig. 7C-E). Since bafilomycin A1 does not induce CASM ⁹³ but disturbs luminal pH, we conclude that it is the less acidic luminal pH of the endolysosmal organelles that is sufficient to interfere with the proper sorting of GLUT1.

The above experiments indicate the role of acidification and integrity of endolysosomal compartments in GLUT1 sorting. Membrane atg8ylation is important for lysosomal repair and homeostasis with specific downstream mechanisms. These processes include: (i) ATG2A, which transfers lipids during lysosomal repair ⁸⁵ and is engaged on damaged lysosomes upon membrane atg8ylation by LC3A ⁹³, and (ii) ESCRT-based lysosomal membrane repair ^80,96,97,116, with membrane atg8ylation specifically recruiting an ESCRT regulator ALG2 ⁹² and ESCRT-I component VPS37A ²⁰. Hence, we tested whether these established systems, involved in maintenance of lysosomal membrane integrity, represented by ATG2 and VPS37, can explain the mechanism underlying the observed effects of membrane atg8ylation on retromer-dependent trafficking. When U2OS ATG2A/ATG2B double KO cells were compared to parental U2OS WT cells, we observed aberrant GLUT1 trafficking, presented as accumulation of cytoplasmic GLUT1 puncta and increased colocalization of GLUT1 with lysosomes (LAMP2A) (Figs. 8A-C and S8A). A similar effect was observed when the previously characterized VPS37A knockout cells ²⁰ were tested (Figs. 8D-F and S8B). These effects were detected with or without induced lysosomal damage (Fig. 8A-F). We interpret these data as evidence that endolysosomal homeostasis and functionality, which is maintained by membrane atg8ylation at all times even under basal conditions, contributes to proper function of retromer-dependent sorting of its cognate cargo such as GLUT1 (Fig. 8E).

Finally, we addressed the strength of effects on GLUT1 trafficking observed with ATG5 KO vs. KOs in other membrane atg8ylation genes, which were significant in all experiments but presented effects lower in magnitude than ATG5 KO (Figs. 4 and 6). One possibility was that since ATG3, ATG5, and ATG7 all participate in the same process of membrane atg8ylation, that our KO mutants had unequal gene inactivation levels. This was ruled out by comparing effects of ATG3 KO, ATG5 KO and ATG7 KO cells for their effects on LC3 puncta formation in a well-defined system of starvation-induced autophagy. The results of these analyses indicated that all three KOs had indistinguishable effects on LC3 puncta formation during canonical autophagy induction by starvation (Fig. S8C,D). Thus, the stronger effects of ATG5 KO on retromer vis-à-vis KOs in other atg8ylation genes reflected additional action of ATG5. In the absence of ATG5, less VPS26 and VPS35 could be pulled down in co-IPs with YFP-VPS29 (Fig. S8E-H). We conclude that ATG5, in addition to contributing to the retromer-dependent sorting via membrane atg8ylation and its downstream effector mechanisms which maintain healthy endolysosomal organelles, has additional effects on the retromer.