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Spatio-temporal processes in autophagosome-lysosome fusion

  • Shizuo Liu , Huan Yan , Jiajie Diao EMAIL logo , Shen Zhang ORCID logo EMAIL logo and Qing Zhong ORCID logo EMAIL logo
Published/Copyright: June 19, 2025
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Medical Review
From the journal Medical Review Volume 5 Issue 4

Abstract

Macroautophagy/autophagy is a lysosome-dependent degradation process involved in cellular energy metabolism, recycling and quality control. Autophagy is a highly dynamic and precisely regulated process, which contains four major steps: autophagic membrane initiation and cargo recognition, autophagosome formation, autophagosome-lysosome fusion and lysosomal degradation. During the terminal phase of autophagy, the merging of the autophagosome and lysosome membranes is critical for the effective breakdown of sequestered cargoes. However, the participated molecules and the interplay among them have not been fully uncovered. The spatiotemporal property of these molecules is crucial for maintaining the orderly fusion of autophagosomes and lysosomes, otherwise it may lead to fusion disorders. In this article, we tend to summarize the molecules mediating autophagosome-lysosome fusion into two categories: effector molecules and regulatory molecules. The effector molecules are soluble N-ethylmaleimide–sensitive factor attachment protein receptor and tethering proteins, and the latter category contains phosphatidylinositol, Rab GTPases and ATG8-family proteins. The spatio-temporal properties of these autophagosome-lysosome fusion mediating molecules will be featured in this review.

Keywords: autophagosome-lysosome fusion; sensitive factor attachment protein receptor; syntaxin 17; autophagy

Introduction

Macroautophagy (hereafter called autophagy) is a conserved intracellular degradation process that involves the formation of a double-membrane vesicle, the autophagosomes, which engulf cellular cargo and then fuse with lysosomes, ultimately leading to cargo degradation by autolysosomes/lysosomes [1]. The transition from omegasomes to autolysosomes engages a substantial ensemble of molecular components, and it is intriguing to explore the rationale behind the systematic progression of this cellular process. Which signal emanating from the sealed autophagosome facilitates the fusion event? And in what sequential manner do the fusion mediators operate upon both the autophagosome and lysosome? In order to better understand the mechanism, we have established a tripartite framework for the regulation of autophagosome-lysosome fusion. At the first step, sensing molecules come into play. Here refers to the detectors of membrane curvature sensors, or pH sensors, which perceiving the closed state of the autophagosome as well as the ambient pH levels, then they relay this information to the secondary level molecules in a real-time mechanism [2], [3]. The secondary level molecules consist of intermediary molecules that bridge the gap between sensing and the subsequent fusion process. Intermediary molecules are normally positioned on autophagosomes but not directly executing fusion event, such as phosphatidylinositol, ATG8-family proteins (ATG8s), Rab GTPases, etc. They always function as molecular switches to ‘turn on’ or ‘turn off’ upon the regulation of effector molecules, then directly or indirectly communicate signals to third-level molecules [4]. The third level molecules are effector molecules like sensitive factor attachment protein receptor (SNARE), tethering factor, etc., which will be recruited to the autophagosome membrane by anchoring to intermediary molecules [5]. Based on the acting order and function, we categorize the first and second level molecules as regulatory molecules and the third level molecules as effectors. Previous reviews have summarized diseases caused by defects in the molecular structure of fusion proteins or related proteins comprehensively [4], [6], [7]. Therefore, in this paper, we focus mainly on the spatiotemporal process of effector molecules in fusion and the role of regulatory molecules on how to maintain the ordering of effector molecules (Figure 1).

Figure 1: The process of autophagosome-lysosome fusion. a) Autophagosomes surrounding organelles and lysosomes localized at the cell periphery move through the cytoskeleton to the vicinity of the perinuclear area. b) Autophagosomes and lysosomes clustered at the cell periphery approach each other in the presence of tethering factors. c) When the two are sufficiently close together, autophagosomes and lysosomal membrane-localized SNARE proteins interact with each other to form a helical complex, which further pulls them in distance until fusion. We have tabulated the key signals for lysosomal and autophagosomal translocation to the perinucleus, and for the localization of tethering factors and SNARE proteins to the corresponding membranes.
Figure 1:

The process of autophagosome-lysosome fusion. a) Autophagosomes surrounding organelles and lysosomes localized at the cell periphery move through the cytoskeleton to the vicinity of the perinuclear area. b) Autophagosomes and lysosomes clustered at the cell periphery approach each other in the presence of tethering factors. c) When the two are sufficiently close together, autophagosomes and lysosomal membrane-localized SNARE proteins interact with each other to form a helical complex, which further pulls them in distance until fusion. We have tabulated the key signals for lysosomal and autophagosomal translocation to the perinucleus, and for the localization of tethering factors and SNARE proteins to the corresponding membranes.

Effector molecules mediating autophagosome-lysosome fusion

SNARE complex

The autophagosome-lysosome fusion process is dependent on SNARE complexes or SNARE-like function complexes, which play a crucial role in lowering the energy threshold required for the merging of membranes. In mammals, four groups of complexes have been identified for autophagosome-lysosome fusion: syntaxin (STX) 17-SNAP29-VAMP8/VAMP7, YKT6-SNAP29-STX7 [8], [9]. STX17-SNAP47-VAMP7/8 in the basal state and LAMP2B-ATG14-VAMP8 in cardiomyocytes [10], [11].

STX17-SNAP29-VAMP7/8

The STX17-SNAP29-VAMP7/8 complex was the first autophagosome-lysosome fusion complex discovered, in which STX17 and VAMP7/8 each contributes one helix and SNAP29 contributes two helices [12]. The recruitment, polymerization and depolymerization of the four-helix complex are delicately regulated respectively.

Release and recruitment of STX17

STX17 is dynamically distributed in the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, ER/Mito contact sites and cytoplasm [12]. Free STX17 in the cytoplasm is in an autoinhibited conformation, with intramolecular interactions between its Habc structural domain and SNARE motif to prevent binding to other SNARE proteins [13]. Under basal conditions, unphosphorylated STING associates to STX17 at the ER, thereby restricting the availability of STX17. However, upon energetic stress, phosphorylated STING loses its binding ability for STX17, allowing STX17 to release from the ER to complete autophagosomes (Figure 2A) [14]. Free STX17 binds to immune-related GTPase M and ATG8 family proteins to form a trimeric complex known as the autophagosome recognition particle, which is transported to the autophagosome [15]. In addition, other proteins are involved in the transport process of STX17, as STX17 is still recruited to the autophagosome membrane after deletion of ATG8s [16], [17]. Upon STX17 approaching to the autophagosome, the positively charged lysines and arginines at the C-terminus of STX17 are drawn to the negatively charged PtdIns4P situated on the autophagosome membrane [18], resulting in STX17’s initial anchoring to the autophagosomal surface (Figure 2B). However, it appears that electrostatic attractions are not as potent as direct intermolecular interactions. The ATG8s associated protein filamentous protein A (FLNA) provides further immobilization of STX17 to autophagosomes, which requires ULK1 (ULK1, a serine/threonine protein kinase involved in multiple processes of autophagy)-mediated phosphorylation on STX17 [19]. It is noteworthy that STX17 has been identified as a key player in the initiation of autophagy, the formation of hybrid pre-autophagosomal structures (HyPAS, formed by fusion of cis-golgi FIP200 vesicles with endosomal-derived ATG16L1 membranes), and the fusion of autophagosomes [14], [20], [21]. The precise signals that regulate the orderly involvement of STX17 in these processes remain unclear. However, they may be related to the different distribution of STX17 or to different binding partners. For example, phosphorylation of S202 of STX17 on the Golgi apparatus facilitates its translocation to the mammalian pre-autophagosomal structure [20]. Phosphorylated STX17 on S202 facilitates it to bind to SERCA2 and E-SYT2 at the ER, which promotes the merging of the endosome with the cis-Golgi to form HyPAS [21]. On the contrary, STING association with STX17 is going to be impaired upon STX17 phosphorylation on S202, which enhances the release of STX17 for recruitment to mature autophagosomes [14].

Figure 2: Effector molecule-mediated autophagosome-lysosome fusion. A, B: autophagic translocation of STX17. STX17 is initially immobilized by STING distributed on the ER and released upon energetic stress. STX17 remains in an autoinhibitory conformation until it binds to SNAP29, the Habc structural domain being able to sequester SNARE. B: Free STX17 forms an ARP and is transported to the vicinity of the autophagosome membrane. The positively charged lysine and arginine at the C-terminus of STX17 are attracted to the negatively charged PtdIns4P localized on the autophagosome membrane. C: Four sets of complexes mediating autophagosome fusion. C-Ⅰ: STX17 distributes to the autophagosome membrane and first participates in the priming complex with SNAP29-YKT6. The helical structural domain of ATG14 also binds to the SNARE structural domain of STX17. C-Ⅱ: Upon proximity of the autophagosome to the lysosome, YKT6 is displaced by VAMP7/8, resulting in the assembly of STX17- SNAP29-VAMP7/8. Displaced YKT6 participates in the formation of YKT6-SNAP29-STX7. C-Ⅲ, Ⅳ: Under basal conditions, SNAP29 is O-GlcNAc-modified and unable to bind to STX17. Positively charged SNAP47 is attracted to the negatively charged PtdIns(4,5)P2 on the phagophore membrane and binds to the SNARE structural domain of STX17. STX17-SNAP47-VAMP7/8 is formed when the membranes are in close proximity to each other. C-Ⅴ, Ⅵ: LAMP2B-ATG14-VAMP7/8 mediates autophagosome fusion in cardiomyocytes. D: Depolymerization of the SNARE complex. STX17-SNAP29-VAMP7/8 is initially immobilized by α-SNAP and then binds to NSF. NSF’s rotational motion causes the SNARE complex to unhelix. E: STX17 condensed vesicles bud from autolysosomes with SNX4 and SNX5. SNX17 acts as a linker to connect SNX4-SNX5 to dynamin-dynamics. SNX4 and SNX5 are then disassembled from the vesicle. STX, syntaxin; SNARE, sensitive factor attachment protein receptor; SNX, sorting nexin.
Figure 2:

Effector molecule-mediated autophagosome-lysosome fusion. A, B: autophagic translocation of STX17. STX17 is initially immobilized by STING distributed on the ER and released upon energetic stress. STX17 remains in an autoinhibitory conformation until it binds to SNAP29, the Habc structural domain being able to sequester SNARE. B: Free STX17 forms an ARP and is transported to the vicinity of the autophagosome membrane. The positively charged lysine and arginine at the C-terminus of STX17 are attracted to the negatively charged PtdIns4P localized on the autophagosome membrane. C: Four sets of complexes mediating autophagosome fusion. C-Ⅰ: STX17 distributes to the autophagosome membrane and first participates in the priming complex with SNAP29-YKT6. The helical structural domain of ATG14 also binds to the SNARE structural domain of STX17. C-Ⅱ: Upon proximity of the autophagosome to the lysosome, YKT6 is displaced by VAMP7/8, resulting in the assembly of STX17- SNAP29-VAMP7/8. Displaced YKT6 participates in the formation of YKT6-SNAP29-STX7. C-Ⅲ, Ⅳ: Under basal conditions, SNAP29 is O-GlcNAc-modified and unable to bind to STX17. Positively charged SNAP47 is attracted to the negatively charged PtdIns(4,5)P2 on the phagophore membrane and binds to the SNARE structural domain of STX17. STX17-SNAP47-VAMP7/8 is formed when the membranes are in close proximity to each other. C-Ⅴ, Ⅵ: LAMP2B-ATG14-VAMP7/8 mediates autophagosome fusion in cardiomyocytes. D: Depolymerization of the SNARE complex. STX17-SNAP29-VAMP7/8 is initially immobilized by α-SNAP and then binds to NSF. NSF’s rotational motion causes the SNARE complex to unhelix. E: STX17 condensed vesicles bud from autolysosomes with SNX4 and SNX5. SNX17 acts as a linker to connect SNX4-SNX5 to dynamin-dynamics. SNX4 and SNX5 are then disassembled from the vesicle. STX, syntaxin; SNARE, sensitive factor attachment protein receptor; SNX, sorting nexin.

Polymerization of STX17-SNAP29-VAMP7/8

The polymerization of STX17-SNAP29-VAMP7/8 occurs through a two-step: 1) STX17 Unlock. 2) assembly of the SNARE complex.

  1. STX17 Unlock. The two tandem transmembrane structural domains of STX17 are required to form a hairpin structure enabling it stably anchored to the autophagosome membrane. However, SNAP29 binds to other SNARE proteins due to lack of neither transmembrane domain nor membrane targeting motif. Vesicular protein sorting 33A (VPS33A) is a Sec1/Munc18 (SM) protein that mediates intracellular vesicle fusion and is a component of the tethered protein homotypic fusion and protein sorting (HOPS) [22], which could clasp autophagic SNAREpins by interacting with STX17. The distribution of STX17 to the autophagosome membrane accelerates the recruitment of the HOPS [23]. In turn, VPS33A acts as a switch that binds to the Habc structural domain of STX17 to maintain the closed state of the SNARE [22]. It is noteworthy that while VPS33A is recruited by STX17, in contrast to the no-go effect on STX17 by VPS33A, gamma-aminobutyric-acid-type-A-receptor-associated protein (GABARAP) presented on the autophagosome is able to compete with the Habc structural domain for binding to the SNARE and mediate the opening of the STX17 conformation [13]. The seemingly contradictory roles of these two molecules reflect the fine regulation of the fusion machinery. Since membrane fusion is driven by a thermodynamically spontaneous protein folding process, these regulatory factors prevent excessive fusion. Additionally, regulatory proteins such as VPS33A can perform sequential inhibition and activation, keeping the complex in a ‘locked’ state that requires only a small triggering stimulus to burst forward [13], [22]. VPS33A and GABARAP are typical examples of regulatory mechanism that can keep the rapid fusion process accurate when triggered.

  2. Assembly of SNAREpins. When the membranes are at a certain proximity, Ser2 on STX17 is dephosphorylated to release VPS33A and the SNARE structural domain of STX17 becomes opened [22]. The deacetylase HDAC2 binds to and deacetylates the SNARE of STX17, allowing STX17 to bind free SNAP29, forming the STX17-SNAP29 binary t-SNARE complex [23], [24]. STX17-SNAP29 will form a priming complex with YKT6 by interacting with SNARE domain on STX17 before engaging VAMP7/8 [25]. This provides a structural basis for the STX17-SNAP29-VAMP8 ternary t-SNARE complex and prevents other proteins from interfering with the formation of the fusion complex (Figure 2C-Ⅰ) [26].

VAMP8 is initially located at the plasma membrane, then RAB21 is activated by its guanine nucleotide exchange factor (GEF) MTMR13 to transport VAMP8 to the lysosomal surface by endocytosis [27]. The stability of SNARE complex can also be regulated. Inhibition of mechanistic target of rapamycin (mTOR) decreases the phosphorylation of VAMP8, which enhances the activity of the SM-like protein sec1 family domain containing 1 (SCFD1) [28], leading to stabilization of the STX17-SNAP29-VAMP8 complex. After autophagosomes approach lysosomes by the help of tethering factors such as HOPS or ATG14, YKT6 will be displaced by VAMP8 to form the ternary STX17-SNAP29-VAMP8 t-SNARE complex, and regulatory proteins like VPS33A [22] or SCFD1 [24], [28] help to maintain the stable conformation of STX17-SNAP29-VAMP8 SNARE complex (Figure 2C-Ⅰ). In addition, ATG14 also functions to stabilize the binary t-SNARE complex STX17-SNAP29 [29]. Unlike the loose affinity between STX17 and YKT6, the sites R45, E52, S55, E56 and K59 in VAMP8 form five hydrogen bonds with S202, H206, N213, N213 and E216 in STX17 respectively [26]. And the conservative ionic layer of the SNARE complex is made up with Q196 in STX17, Q84 and Q230 in SNAP29 and R37 in VAMP8 [30]. By these intermolecular interactions, the zippering model of SNARE complex is tightly maintained (Figure 2C-Ⅱ). As another lysosome-resident R-SNARE, VAMP7 is also directly involved in fusion, while it shows up for two variants as VAMP7A or VAMP7B. VAMP7A facilitates membrane fusion by interaction with STX17, yet VAMP7B plays an inhibitory role by association with STX17, which is regulated by a VAMP7B binding protein divergent protein kinase domain 2A (DIPK2A)/DIA1 [31].

Dissociation and recycling of SNARE complex

The highly conserved hexametric AAA-ATPase N-ethylmaleimide sensitive factor (NSF) and the cofactor alpha-soluble NSF attachment protein (αSNAP) are crucial for SNARE depolymerization [32], [33], which can be divided into three steps: At first, the reverse-parallel αSNAP binds to the trans-autophagic SNARE complex [34], and this binding may occur via electrostatic forces on the surface of the autophagic SNARE complex [13]. αSNAP acts as an articulating protein, providing a firm grip between the SNARE complex and NSF and promoting depolymerization of the SNARE complex. Secondly, the αSNAP-SNARE complex binds to NSF to form a 20S complex. Finally, in the ATP-bound state, the D1 structural domain of NSF undergoes a rotational movement and the SNARE complex is then disassembled (Figure 2D) [33].

NSF/αSNAP locate on the outer membrane of autolysosomes and deficit of αSNAP inhibits autophagic flux [34]. This may due to the insufficient depolymerization of SNARE complexes on autolysosomes resulting in shortage of monomeric SNARE proteins to mediate new cycles of autophagosome-lysosome membrane fusion. The depolymerized monomers of SNARE proteins are located on the surface of autolysosomes. Rab32 and Rab38 then mediate the formation of the heterodimer sorting nexin 4 (SNX4)-SNX5 [35], [36]. This heterodimer recognizes STX17 on autolysosomes and buds off to create STX17-positive vesicles. SNX17 serves as a linker for SNX4-SNX5-STX17 and dynactin-dynein, facilitating STX17 recycling (Figure 2E) [36].

STX17-SNAP29-VAMP7/8 is the predominant and most studied SNARE complex for autophagosome-lysosome fusion. The complex involves various structural changes during fusion; however, the spatiotemporal regulation of the process remains unclear. The unique dual transmembrane structure of STX17 slows fusion by increasing protein-lipid mismatches [37]. However, proteins and NSF/α-SNAP are in an antagonistic state during membrane fusion process such as vesicles: SM proteins act as highly dynamic templates to mediate the assembly of SNARE complexes, while NSF/α-SNAP mediates the disassembly of SNARE complexes [38], [39]. The crosstalk between SM proteins and NSF/α-SNAP in autophagy remains unclear. Moreover, α-SNAP plays roles not only in the degradation of autophagic SNARE complexes, but also in endocytosis and other intracellular trafficking processes [34], which left the question still unanswered on how the SNARE complex is regulated to be degraded.

YKT6-SNAP29-STX7

YKT6 is a conserved R-SNARE protein in eukaryotes. YKT6-SNAP29-STX7 was previously found to mediate autophagy-lysosomal fusion independently of STX17-SNAP29-VAMP7/8 [25], but recent studies have shown that YKT6 also promotes the assembly of STX17-SNAP29-VAMP7/8 [26]. YKT6 is consisted by an N-terminal longin structural domain, a SNARE domain, and a C-terminal “CCXIM” motif. To prevent premature binding of YKT6 to SNARE proteins, YKT6 exhibits a closed conformation. When YKT6 is not engaged in autophagosome or vesicle fusion, it is dispersed throughout the cytoplasm. Mechanistically, ULK1 maintains YKT6 in an inactive state by phosphorylating Thr156, causing the Longin domain to fold back onto the SNARE domain, effectively closing the SNARE complex [40], [41]. Upon autophagy induction, YKT6 can be phosphorylated by a Ca2+-dependent phosphatase calcineurin (CaN) prior to transport to the autophagosome surface. Ca2+-dependent phosphorylation provides a negative charge at YKT6 Ser174 and disrupts the hydrophobic interaction between the Longin and SNARE structural domains, facilitating the transition to an open conformation (Figure 2C-Ⅰ, Ⅱ) [42]. Consequently, the open conformation of YKT6 facilitates the targeting of the Longin domain to the autophagic membrane, and the exposure of the SNARE domain by YKT6, bearing a higher affinity to STX17, allows YKT6 to preferentially form a priming complex with STX17-SNAP29. Once VAMP8 displaces YKT6 from YKT6-SNAP29-STX17, YKT6 immediately forms the STX7-SNAP29-YKT6 complex with lysosome-localized STX7 [26]. Thus, YKT6 can interact with two sets of SNARE helical complexes in succession and increase the efficiency of membrane fusion.

The interaction of YKT6 with these complexes suggests that YKT6 is subject to precise spatio-temporal regulation. Presumably from YKT6 switching in different complexes, it has a higher affinity for STX17 than for STX7, and VAMP7/8 has a higher affinity for STX17-SNAP29 than for YKT6 [26]. Variations in phosphorylation sites of YKT6 may mediate the interaction of YKT6 with different molecules. Wang’s group and Caraveo’s group showed that Ca2+-mediated phosphorylation of mammalian YKT6 Ser174 could turn on its open conformation to promote autophagosome-lysosome fusion [41], [42], but Kraft’s group showed that ULK1-phosphorylated Thr156 on YKT6 inhibited fusion as opposite [43]. This suggests that different phosphorylation sites of YKT6 may be the switch for the spatio-temporal regulation of YKT6 conformational closure.

Considering the recruitment mechanism, there is evidence showed that Yeast Ykt6 is localized to the ER, vesicles and endosomes, and is recruited to the autophagosome by the Dsl1 complex (Dsl1, Dsl3 and Tip20) and COPII-encapsulated vesicles upon autophagy induction [40]. However, how YKT6 is recruited onto autophagic structures remains unknown in mammals. Neither phosphorylation, farnesylation, norpalmitoylation, affects the autophagosomal localization of YKT6, which have previously been implicated in the membrane localization of YKT6 [25], [43]. Given that YKT6 can also mediate other types of membrane fusion, there may be uncovered specific activation signal to mediate autophagosomal membrane localization of YKT6. Furthermore, the importance of priming complex formation needs to be further investigated. In addition to promoting the assembly of STX17-SNAP29-VAMP7/8, YKT6 also recruits VPS33A by its Longin domain in Drosophila [44]. It is interesting to figure out whether the priming complex could also recruit other regulatory proteins to facilitate subsequent fusion.

STX17-SNAP47-VAMP7/8

STX17-SNAP47-VAMP7/8 is a group of four-helix complexes that mediate quality-controlled autophagy in the basal state [10]. SNAP47 was initially found to be distributed on the plasma membrane surface of neurons to mediate vesicle fusion [45]. In other tissues, SNAP47 is normally localized to the mitochondria. SNAP47 initially binds to ATG8s and is transported around the phagosome. Its positively charged amino acids are then drawn to the negatively charged PtdIns(4,5)P2 localized on the phagosome, leading to SNAP47’s recruitment to the phagosome. This ultimately results in the formation of the STX17-SNAP47-VAMP7/8 complex. (Figure 2C-Ⅲ, Ⅳ) [10]. It is worth noting that fusion systems in autophagy under different contexts can be classified into different categories by their labor. When basal autophagy occurs, O-GlcNAcylation inactivates SNAP29 to prevent its participation in the assembly of the SNARE complex, facilitating the binding of SNAP47 to STX17 [46]. One of the possible explanations is, STX17 only localizes to the autophagosome upon closure of autophagosome membrane, so that SNAP 47, pre-localized to the phagosome, may be more favorable of binding to STX17 than free SNAP29. Additionally, prior to the approach of autophagosomes and lysosomes toward each other, ATP13A2 (A lysosomal transmembrane P5B-type ATPase) localizes to the lysosome to recruit HDAC6. HDAC6 is deacetylated only under basal autophagy conditions, and this recruitment sets the stage for assembling the F-actin network, which facilitates the convergence of autophagosomes and lysosomes. [47], [48].

Interestingly, it seems that basal autophagy employs unique fusion complexes and regulatory molecules from starvation-induced autophagy. One of the examples is that PACSIN1 specifically promotes basal-conditioned amphiphage-autophagosome fusion. Moreover, the fusion partner seems to be different. In basal autophagy, autophagosomes preferentially fuse with endosomes to form amphiphages and then with lysosomes. Yet in starvation-induced autophagy, autophagosomes often fuse directly with lysosomes [49].

LAMP2B-ATG14-VAMP8

LAMP2B-ATG14-VAMP8 is a unique set of fusion complexes in cardiomyocytes. LAMP2B (a lysosomal resident protein) deficiency is the major cause of Danon disease and has recently been linked to autophagy [50]. Upon autophagy stimulation, LAMP2B localizes to autophagosomes and its C-terminal coiled-coil domain (CCD) structural domain binds to ATG14 and VAMP8 to mediate autophagosome-lysosome fusion (Figure 2C-Ⅴ, Ⅵ) [11]. Liu et al. showed that LAMP2B is regulated by cellular repressor of E1A-stimulated genes 1 during the fusion process [51], but the exact regulatory mechanism remains unclear.

Notably, ATG14, an important component of class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), was first found to be involved in autophagy initiation and has now been shown to be an important molecule in autophagosome-lysosome fusion as well. Mammalian ATG14 has two isoforms: long (492 aa) and short (379 aa) splice isoforms, designated ATG14L and ATG14S [52]. The different isoforms may be involved in different autophagic processes: In contrast to ATG14L, ATG14S lacks the first 113 amino acids at the N-terminus, which contain a conserved cysteine repeat structural domain. This domain is essential for mediating the fusion of autolysosomes, whereas it is dispensable for binding BECN1 (a component of PI3KC3-C1) [29], [52]. The mitochondrial outer membrane protein Miga is able to recruit ATG14 to mitochondria and also has a function in stabilizing ATG14 and Drosophila Syx17, but the exact mechanism of ATG14 transport to the autophagosome remains unclear. ATG14L is transported to the vicinity of mature autophagosomes. Under the localization of PtdIns4P, the helical structural domain of ATG14 binds to the SANRE structural domain of STX17 [53], [54]. This binding may have similarities to YKT6-STX17-SNAP29: formation of an intermediate helical complex to stabilize STX17-SNAP29 [29]. Two molecules have been found to negatively regulate ATG14L. One is RUNDC1, which stimulates homo-oligomerisation of ATG14 to inhibit ATG14 dissociation and block VAMP8 translocation [55]. The other is MARCH7, which mediates mixed polyubiquitination to reduce ATG14 solubility [56].

Tethering factors

Tethering factors mediate the proximity of autophagosomes and lysosomes to each other and participate in fusion in concert with SNAREs. Most of the tethering factors act in a similar way, being anchored by Rab GTPases, ATG8s or phosphatidylinositol that is recruited to the surface of autophagosomes or lysosomes. Interestingly, tethering factors appear to work as an integrated machine. At least seven tethering factors have been identified (HOPS complex, ATG14, PLEKHM1, EPG5, Tectonin β-propeller repeat containing 1[TECPR1], GRASP55, ATG8s), but why does the autophagosome show a significant decrease in tethering activity when either of them is knocked down? The specific structural and spatiotemporal properties of each tethering protein may be one of the reasons. In the following section, we will introduce the seven tethering proteins discovered so far.

HOPS complex

Recruitment of HOPS subunits

HOPS complex is a conserved tethered complex consisting of VPS11, VPS16, VPS18, VPS33, VPS39 and VPS41 [57]. In the “hook-up” model proposed by Zhang et al., HOPS may initially assemble in the cytoplasm from two subcomplexes (quaternary HOPS (VPS33A-VPS16-VPS18-VPS41) and binary HOPS (VPS39-VPS11) and respectively recruited to autophagosomes and lysosomes [58]. Each subcomplex requires an anchoring site to fix them to the membrane: autophagosome-localized Rab2 and lysosome-localized Rab39A. Rab2 is initially located in the Golgi apparatus, on which it is activated. Upon activation, it is encapsulated in vesicles and translocated to the autophagosome via microtubules. Once on the autophagosome, it interacts with RUBCNL/Pacer and STX17 to stabilize the membrane surface [59]. Rab39A undergoes prenylation and is activated by C9orf72, localizing to lysosomes [58]. Upon translocation of Rab proteins onto the target membrane, the HOPS complex is able to be recruited and assembled.

Assembly of HOPS complex

How the HOPS complex is assembled remains unclear. Previous studies have shown that both VPS33A-VPS16 and VPS18-VPS41 interact with each other via specific structures [60], [61], and based on the precision of autophagosome fusion, we speculate that HOPS subunits may in a two-by-two arrangement before localizing to the corresponding membrane structure. VPS18 and VPS41 bind together to form stable heterodimers that are recruited to the autophagosome membrane by the Rab2-STX17-Pacer complex [60]. Subsequently, VPS16 may binds to the SM protein VPS33A and binds to VPS18-VPS41 to form the tetrameric HOPS complex [61].

Regulation of the HOPS complex

Three binary HOPS (VPS39-VPS11) recruitment proteins have been identified, and one of them is the Rab7 effector protein PLEKHM1, which binds to the Rab GTPase Arl1b and Rab7. PLEKHM1 acts as a signaling platform to recruit VPS39-VPS11 [62]. Another VPS39-VPS11 recruiter is the BLOC-1-related complex (BORC) [63]. The third one is Rab39A, as previously mentioned (Figure 3A). The precise manner of interaction between the three is currently unclear.

Figure 3: Tethering proteins in autophagosome-lysosome fusion. A. Quaternary HOPS (VPS33A-VPS16-VPS18-VPS41) and binary HOPS (VPS39-VPS11) and are anchored to Rab2 on the autophagosome and Rab39A on the lysosome, respectively. Then, assembled into complete HOPS complexes. B. GRASP55 is anchored to autophagosomes and lysosomes via LC3 and LAMP2, respectively. C. TECPR1 competes with ATG16 for ATG5-ATG12. On autophagosomes, ATG5-ATG12 is involved in TECPR1-anchoring with PI3P. Rab7 is involved in lysosomal end anchoring of LAMP2. D. PLEKHM1 initially binds to Rab7 on the endosome. Upon conversion of PtdIns4P to PtdIns(4,5)P2 on endosomes, PLEKHM1 is released by Rab7. Then, PLEKHM1 may bind to Arl8b on lysosomes. TRIM22 promotes the binding of PLEKHM1 to GABARAPs on autophagosomes. E. ATG14 and ATG8s participate in tethering. F. EPG5 first binds to TGM2. LC3, WDR45 and PI3P are involved in the anchoring of EPG5 at the autophagosome end. Rab7, VAMP8, WDR45B and PI3P are involved in the anchoring at the lysosome end. HOPS, homotypic fusion and protein sorting; TECPR1, tectonin β-propeller repeat containing 1; TGM2, Transglutaminase 2; GABARAP, gamma-aminobutyric-acid-type-A-receptor-associated protein.
Figure 3:

Tethering proteins in autophagosome-lysosome fusion. A. Quaternary HOPS (VPS33A-VPS16-VPS18-VPS41) and binary HOPS (VPS39-VPS11) and are anchored to Rab2 on the autophagosome and Rab39A on the lysosome, respectively. Then, assembled into complete HOPS complexes. B. GRASP55 is anchored to autophagosomes and lysosomes via LC3 and LAMP2, respectively. C. TECPR1 competes with ATG16 for ATG5-ATG12. On autophagosomes, ATG5-ATG12 is involved in TECPR1-anchoring with PI3P. Rab7 is involved in lysosomal end anchoring of LAMP2. D. PLEKHM1 initially binds to Rab7 on the endosome. Upon conversion of PtdIns4P to PtdIns(4,5)P2 on endosomes, PLEKHM1 is released by Rab7. Then, PLEKHM1 may bind to Arl8b on lysosomes. TRIM22 promotes the binding of PLEKHM1 to GABARAPs on autophagosomes. E. ATG14 and ATG8s participate in tethering. F. EPG5 first binds to TGM2. LC3, WDR45 and PI3P are involved in the anchoring of EPG5 at the autophagosome end. Rab7, VAMP8, WDR45B and PI3P are involved in the anchoring at the lysosome end. HOPS, homotypic fusion and protein sorting; TECPR1, tectonin β-propeller repeat containing 1; TGM2, Transglutaminase 2; GABARAP, gamma-aminobutyric-acid-type-A-receptor-associated protein.

The working mechanism of HOPS

HOPS has two distinct interfaces. One interface is with Rab proteins, which serve to anchor both ends of HOPS to autophagosomes and lysosomes. The other interface interacts with SNARE via SM proteins, employing a specific mechanism as previously discussed. The resolution of the yeast HOPS complex structure by cryo-electron microscopy revealed that HOPS has a triangular structure [64]. Given the conserved nature of membrane fusion and the findings of recent studies [58], [65], [66], a hypothesis was formed that the three corners of the triangle formed by VPS41, VPS39, and VPS33A-VPS16 might be bound to Rab39A, Rab2, and Stx17. VPS11 and VPS18 act as the backbone, stabilizing the complex. The HOPS complex displays a certain degree of flexibility, as evidenced by its capacity to contract or elongate [67]. VPS41 and VPS39 are capable of oscillating between 10° and 20° [64], which suggests that the movement of HOPS and the interaction of SNAREs can be stabilized during the mutual proximity of membranes. Furthermore, SNARE complexes exhibit a high energy requirement for fusion, and HOPS is capable of reducing the energy barrier to fusion by relying on membrane curvature stress through its own triangular structure, in addition to stabilizing SNARE complexes [64], [68], [69].

GRASP55

GRASP55 is a tethering factor residing on the Golgi that senses intracellular energy changes. Upon starvation, decreased GRASP55 O-GlcNAcylation induces its translocation to autophagosomes and lysosomes. Attachment of GRASP55 on autophagosomes dependents on LC3-II anchoring, while the localization on late endosomes/lysosomes of GRASP55 is mediated by LAMP2 interaction [70]. GRASP55 can also enhance RAB7A activity to promote the interaction between RAB7A and HOPS [71]. It is noteworthy that GRASP55 may be trapped during the initial stages of autophagy [72] and released, during autophagosome maturation, closure and fusion stage [71], [73]. This spatiotemporal regulatory mechanism likely ensures that GRASP55 operates precisely at the specific stages of autophagy progression (Figure 3B).

TECPR1 and ATG8s

TECPR1 has a different significance from the tethering factor we described previously, as it not only promotes membrane fusion, but also represents the transitional stage between autophagosome maturation and fusion. Crystal structure analysis showed that both ATG16L1 and TECPR1 have α-helical structures and compete for the ATG5 binding site through the conserved ATG5 (Five)-Interacting Motif (AFIM) sequence. During autophagosome maturation, the AFIM sequence of ATG16L1 binds to Atg12-Atg5 to form the ATG12-ATG5-ATG16L1 complex to mediate the lipidation of ATG8s.

TECPR1 not only contributes to autophagosome maturation by forming the ATG12-ATG5-TECPR1 complex, but also mediates autophagosome-lysosome fusion. In this process, the N-terminal β-propeller (TR1) domain of TECPR1 can co-localize with lysosomal Rab7 and LAMP2 [74], and the C-terminal end can be anchored to the autophagosome membrane by the AIR and pleckstrin homology (PH) domains. TECPR1 is ‘pinned’ to the autophagosome membrane by two ’nails’, namely the ATG12-ATG5 coupling and PtdIns3P affinity (Figure 3C). The N-terminus of TECPR1 is likely to be first anchored to the lysosome and then brought closer to the autophagosome by the action of other tethering factors. The pH level near lysosomal membrane may be lower than that of other cytoplasmic space, increasing the dissociation constants of ATG5 and ATG16L1 and facilitating the binding of ATG5 to TECPR1, resulting in stronger intermolecular forces [75]. The binding of TECPR1 to ATG5 alone assists in exposing specific binding sites on ATG12-ATG5, thereby creating the necessary conditions for the AIR structural domain of TECPR1 to bind to the ATG12-ATG5 coupler. Then, the membrane PI3P-coupled PH domain of TECPR1-ATG12-ATG5 complex exposes [76], [77], and TECPR1 is stably anchored to the autophagosome membrane to exert tethering activity. Therefore, ATG12-ATG5 may function with different binding partners at different stages of autophagy. After autophagosome maturation, competitive binding of TECPR1 terminates the lipolytic activity of ATG12-ATG5-ATG16L1 and unloads ATG16L1, which is eventually converted to tethering activity.

The ATG16L1-ATG12-ATG5 complex not only lipidates ATG8s, but also sets the stage for subsequent membrane tethering and fusion [78]. The lipidated LC3C is distributed on the membrane surface of autophagosomes and mediates the proximity of autophagosomes and lysosomes to each other, which binds to the LC3 interaction region (LIR) sequence at the N-terminal end of TECPR1 (Figure 3E) [74], [79]. Interestingly, it is still not known how LC3C mediates membrane traction and fusion and the structure altering of TECPR1 during the traction process. Although Zhong’s group and Sasakawa’s group showed that TECPR1 binds to PtdIns3P on autophagosomes through the PH structural domain [76], [77], Wollert’s group showed that TECPR1 does not bind to PtdIns3P, but instead binds to lysosomal localized PtdIns4P [74]. Thus, the mechanism of TECPR1 anchoring to lysosomal membrane is still variable depending on cell type or distinct stimulate of autophagy.

PLEKHM1

PLEKHM1 is a Rab7 effector protein that participates the fusion of late endosomes/autophagosomes with lysosomes [80]. Arl8b and Rab7 are two small GTPases, the former being enriched on peripheral lysosomes [62], and the latter on the more acidic, late endosomal/lysosomal pool in the perinuclear region of the cell [81]. Marwaha et al. found that SKIP/ PLEKHM2, which has a RUN structural domain similar to PLEKHM1, binds to Arl8b and interacts with the kinesin-1 motor to transport lysosomes to the periphery of autophagosomes [62]. After lysosomes are translocated to the perinuclear or autophagosomal periphery, PLEKHM1 competes with SKIP for binding to Arl8b and localizes to the lysosomal membrane surface [82]. TRIM22 acts as a bridge for PLEKHM1 to be anchored to the autophagosome membrane. The B-box zinc finger domain at the N-terminal end of TRIM22 binds to PLEKHM1, the SPRY at the C-terminal end binds to GABARAPs, and finally promotes direct binding of PLEKHM1 to GABARAPs [83]. Previous studies have shown that PLEKHM1 has an LIR that binds to ATG8s. In fact, five residues of GABARAPs (G18, K20, R/K47, D54, F/W62) have a stronger binding specificity for PLEKHM1, and the specific charges of these residues make PLEKHM1 more stably anchored to autophagosome (Figure 3D) [84]. Following the anchoring of PLEKHM1 to autophagosomes and lysosomes, two subunits of HOPS, VPS41 and VPS39, are recruited to the RUN structural domain of PLEKHM1 to facilitate the fusion of autophagosomes and lysosomes [85].

An interesting question in the process of PLEKHM1 exerting its tethering function is how PLEKHM1 specifically mediates the fusion between endosomes/autophagosomes and lysosomes. In terms of localization, PLEKHM1 is first anchored to endosomes via Rab7 and then to lysosomes via Arl8b. On the autophagosome, it is anchored via GABARAPs. This means that when autophagy is activated, specific signals can dissociate endosome-localized PLEKHM1 from Rab7 or deprive it of its ability to mediate endosome/lysosome fusion. There are two types of regulatory signals that mediate PLEKHM1 translocation. In the endosomal pool, PtdIns4P is enriched on the Rab7-positive compartment, which can be converted to PtdIns(4,5)P2 upon activation of autophagy to dissociate Rab7 from PLEKHM1. Interestingly, dissociated PLEKHM1 can bind to Arl8b and promote autolysosome formation [86]. In addition, under nutrient-rich conditions, mTOR typically localizes to the lysosomal surface to phosphorylate PLEKHM1 on endosomes. However, during autophagy activation, mTOR detaches from the lysosome, leading to the dephosphorylation of PLEKHM1. This shift in mTOR activity may contribute to the differential regulation between endosomal/autophagosomal and lysosomal fusion [87].

EPG5

EPG5 is a tethering factor that is involved in the fusion of autophagosomes with lysosomes and the assembly of t-SNARE complexes [88]. The overall structure of hEPG5 is reminiscent of a “shepherd’s crook”, comprising a rigid circular “hook” and a flexible “shaft” [89]. The fusion specificity of EPG5 is dependent upon three ‘safe signs’ present on autophagosomes and lysosomes. 1) Transglutaminase 2 (TGM2) recognizes and binds to betaine homocysteine methyltransferase (BHMT) after being transported to the lysosomal surface by flotillin 1 (FLOT1) at the cell membrane [90]. The LIR of TGM2 binds to LC3 on the surface of autophagosomes. Furthermore, the binding of EPG5 to TGM2 and LC3 is also a prerequisite for the assembly of the STX17-SNAP29-VAMP8 complex [91]. 2) EPG5 is localized to lysosomes via Rab7 and VAMP8, and at the other end to autophagosomes via its LIR, which is bound to LC3 [88]. 3) other EPG5-associated proteins identified like WDR45 and WDR45B belong to a family of PtdIns3P-binding proteins containing WD40 repeats, along with WIPI1 and WIPI2. WDR45 and WDR45B are localized to the autophagosome and lysosome via PtdIns3P and can bind to the two ends of EPG5 respectively, resulting in helping the assembly of SNARE proteins-EPG5 fusion machinery (Figure 3F) [92].

In this section, we introduce the effector molecules that mediate autophagosome-lysosome fusion: the SNARE complex and the tethering factor. The ends of the tethering factor are anchored to the autophagosome and lysosome, respectively, bringing the two closer together. When the two are close enough, the assembly of the SNARE complex is able to create a localized tensile force on the membrane, causing it to bend locally at the contact zone. When the distance between the membranes is reduced to the nanometer scale, factors such as electrostatic repulsion and hydrophobic interaction begin to dominate, forcing the two membranes to undergo localized deformation, which facilitates the formation of fusion pore. Exploring the recruitment, assembly or regulation of molecules involved in this process is beneficial to our further understanding of the mechanism of autophagosome-lysosome fusion.

Spatiotemporal regulation of autophagosome-lysosome fusion

Although some progress has been made on the effector molecules of autophagosome-lysosome fusion, the signaling molecules that trigger membrane fusion remain unclear. As can be seen from the preceding description, phosphatidylinositol, Rab GTPases and ATG8s are involved in the transport, recruitment and assembly of SNAREs and tethering proteins. It is also noteworthy that they play an important role in autophagy initiation, maturation, and closure. Consequently, the presence or absence of specific phosphatidylinositols, as well as the activity of Rab GTPases, are typically closely associated with the regulation of autophagosomes at different stages [93], [94], [95]. The following discussion will focus on the spatiotemporal regulation of autophagy, with particular attention paid to main molecules with existing evidence.

The spatiotemporal regulation of autophagosome and lysosome fusion can be divided into two distinct phases. The first phase involves the relocation of both organelles from their respective subcellular compartments to the perinuclear region following the fulfillment of certain fusion prerequisites. The second phase entails the recruitment of effector molecules to the membranes of autophagosomes and lysosomes and their subsequent interaction.

Autophagosomal and lysosomal relocation to the perinuclear region

It is proposed that autophagosomes may only acquire the capability to move to the perinuclear region for fusion with lysosomes once they have attained fusion competency, which is driven by kinesins. Prior to this, FYCO1 forms complexes with Rab7, PtdIns3P and LC3, which are localized on the autophagosome membrane. This enables autophagosome to move along the microtubules in an anterograde transport heading to the cell periphery [96]. Once the autophagosome is closed or the corresponding fusion proteins have been recruited, the phosphorylation of LC3B-T50 by the Hippo kinase STK4/MST1 may dissociates FYCO1 from LC3B [97], [98]. Additionally, the scaffolding protein JIP1 and the dynamin activator dynactin may form the complex with LC3, allowing autophagosomes to retrograde to the perinuclear region (Figure 4) [99].

Figure 4: The in-cell spatial regulation of autophagosome-lysosome fusion. Autophagosomes are initially transported by FYCO1 to move away from the perinuclear area. After fusion conditions are exhibited, STK4 phosphorylates LC3 to dissociate from FYCO1. Movement towards the perinucleus is then mediated by JIP1. Endosomal conversion of PtdIns4P to PtdIns(4,5)P2 promotes PLEKHM1 translocation to the lysosome, allowing lysosomal movement towards the perinucleus. GABARAP may dissociate from VPS37A after autophagosome closure, and then activate PI4KIIα to produce PtdIns4P. PtdIns4P attracts STX17, which recruits thrombospondins and forms the SNARE complex. Upon fusion of autophagosomes with lysosomes, degraded mitochondria expose mitochondrial DNA. MtDNA activates TLR9, which promotes OCRL recruitment to lysosomes. OCRL activates TRPML1 to release Ca2+ and promote autophagosome-lysosome fusion. STX, syntaxin; GABARAP, gamma-aminobutyric-acid-type-A-receptor-associated protein.
Figure 4:

The in-cell spatial regulation of autophagosome-lysosome fusion. Autophagosomes are initially transported by FYCO1 to move away from the perinuclear area. After fusion conditions are exhibited, STK4 phosphorylates LC3 to dissociate from FYCO1. Movement towards the perinucleus is then mediated by JIP1. Endosomal conversion of PtdIns4P to PtdIns(4,5)P2 promotes PLEKHM1 translocation to the lysosome, allowing lysosomal movement towards the perinucleus. GABARAP may dissociate from VPS37A after autophagosome closure, and then activate PI4KIIα to produce PtdIns4P. PtdIns4P attracts STX17, which recruits thrombospondins and forms the SNARE complex. Upon fusion of autophagosomes with lysosomes, degraded mitochondria expose mitochondrial DNA. MtDNA activates TLR9, which promotes OCRL recruitment to lysosomes. OCRL activates TRPML1 to release Ca2+ and promote autophagosome-lysosome fusion. STX, syntaxin; GABARAP, gamma-aminobutyric-acid-type-A-receptor-associated protein.

The distribution of lysosomes is also precisely regulated [100]. Lysosomal membranes are enriched with PtdIns3P when the cell is well nourished, which results in mTOR activation to promote anabolism and FYCO1 recruitment to the lysosomal membrane, to further transport the lysosome to the periphery. Thereby, the lysosome positioning facilitates the passage of growth factors across the plasma membrane, maintaining mTOR activation [101]. In response to starvation, PtdIns4P on endosomes is rapidly converted to PtdIns(4,5)P2, resulting in the dissociation of PLEKHM1 from Rab7 [86]. Rab7 on peripheral lysosomes may re-recruit RILP and PLEKHM1, while the latter competes with SKIP for Arl8b [62]. Binding of PLEKHM1 to Arl8b serves two functions: firstly, for the recruitment of the binary HOPS (VPS39-VPS41) in preparation for the complete assembly of the subsequent HOPS, and secondly, to recruit RILP and transport the lysosomal retrotransport to the perinuclear region [62]. Concurrently, inositol polyphosphate-5-phosphatase E (INPP5E) is transported to the lysosome, where it catalyzes the conversion of PtdIns(3,5)P2 to PtdIns3P. This process stabilizes actin filaments on the lysosome, which may further facilitate lysosomal translocation to the perinucleus [102]. mTOR inhibition results in the dephosphorylation of VAMP8, and multiple lysosomes form clusters by the help of VAMP8, which surround the single autophagosome, to enter the pre-fusion state [103]. Notably, mTOR localized to lysosomes in the periphery inhibits PI4K2A, which promotes the conversion of PtdIns 3P to PtdIns 4P. restores PI4K2A activity when autophagy is induced, allowing lysosomes to enrich for PI4P and translocate to the perinuclear area. However, how it interacts with the cytoskeleton is unclear [104].

Another fascinating question is how the autophagosome translocation is triggered. It has been proposed that the autophagosome-lysosome fusion process occurs in the peripheral cytoplasm, as this is the site of autophagy initiation [5], [63]. However, evidences have been provided that autophagosome and lysosome may delivered to perinuclear region to conduct membrane fusion [105]. In this process, lysosomes are first transported to the perinucleus, as they can receive translocation signals (e.g., mTOR) more readily than mature autophagosomes. Subsequently, lysosomes send signals to allow autophagosomes translocation. For instance, the knockdown of lysosome membrane-resident BORC, which mediates kinesin-dependent movement of lysosome and recruitment of HOPS, results in lysosome aggregating in the perinuclear area and autophagosomes dispersing in the peripheral cytoplasm [63].

Autophagosome-recruiting effector molecules

In the event of lysosomes fusing with unclosed autophagosomes, the harmful lysosomal enzymes are released into the cytoplasmic lysate. The closure and fusion of these membranes are subject to strict spatiotemporal regulation. As an illustration of the fusion of apoptotic cell phagosomes, following the formation of a phagosome, PPK-1 produces PtdIns4P on the phagosome. Both the PtdIns3P phosphatase MTM-1 (to catabolize PtdIns3P) and the class II PI3 kinase PIKI-1 (to produce PtdIns3P) can bind to PtdIns4P, which has double effect. Firstly, the two enzymes work together to regulate the levels of PtdIns3P on the phagosome. Before the phagosome closes, MTM-1 maintains low levels of PtdIns3P. After closure, MTM-1 may dissociate from PtdIns4P, allowing PIKI-1 to reproduce PtdIns3P. Secondly, the two enzymes, combine with PPK-1, the SNX9 family protein LST-4, and PtdIns4P are able to mediate phagosome closure [106]. The signaling regulation of these phosphatidylinositols and the corresponding enzymes is a highly attractive topic. Initially, PtdIns3P is localized at the plasma membrane, where it is degraded after phagosome formation. Following this, it is re-enriched and recruited for protein-mediated membrane fusion after phagosome closure [107]. It is noteworthy that the deletion of PtdIns4P, the phosphatase that binds to it (PIKI-1 or MTM-1), or the early enrichment of PtdIns3P prior to closure can affect phagosome closure [106].

Although molecules capable of linking closure and fusion have not been directly identified in autophagy, recent studies have demonstrated that GABARAP plays a role in autophagosome closure and phosphatidylinositol production [108], [109]. Furthermore, Mizushima’s team demonstrated that PtdIns4P is rapidly enriched in autophagosomes following closure and functions as a mediator of membrane fusion [110]. It is therefore reasonable to hypothesize that the ATG8 family member GABARAP may act as a linker between closure and fusion. During the closure phase, GABARAP binds to the essential ESCRT-I component VSP37A [109]. At this juncture, it is also possible that GABARAP may bind to PI4KIIα; however, the latter activity may be inhibited. Following the closure of the autophagosome, the dissociation of GABARAP from VPS37A restores PI4KIIα activity, allowing it to produce PtdIns4P (Figure 4). It is also possible that GABARAP can recruit PI4KIIα only after dissociation of GABARAP from VPS37A. And recent study showed that the negative surface charge driven by PI4P attracts free STX17 and inserts it into autophagosome membranes [108], [109], [110]. Notably, it is also required that other specific molecules interacting with PtdIns4P, for effectively recruiting STX17. For example, the deficiency of Yeast PtdIns4P phosphatase Sac1 results in the accumulation of PtdIns4P on autophagosome membranes, which subsequently inhibits the recruitment of SNARE proteins [111]. The distribution of STX17 on autophagosome serves as a signaling platform for the sequential recruitment of multiple molecules. Firstly, RUBCNL/Pacer facilitates the anchoring of UVRAG-PI3KC3 sub-complex to STX17, thereby generating PtdIns3P at the autophagosome membrane [112]. Secondly, ATG14 may exhibit a greater affinity for STX17 compared to UVRAG-PI3KC3. Following the binding of UVRAG-PI3KC3 to STX17, ATG14 competes with it for binding STX17, thereby preparing for the autophagosome-lysosome fusion. Subsequently, Rab2A is transported from the Golgi apparatus to the autophagosome, where it forms a complex with Pacer and STX17, thereby facilitating the recruitment of HOPS [59]. Furthermore, Rab33B, a Golgi-resident protein, is translocated to the phagosome upon autophagy induction, and Rab33B-GAP binds to ATG16L and mediates the lipidation of LC3. However, Rab33B is inactivated when the autophagosome matures, and if Rab33B remains in the GAP state, it will inhibit autophagosome-lysosome fusion [113]. It may also function as a closure-fusion linker.

A model of autophagosome-lysosome fusion incorporating positive feedback has been proposed in recent years. Starvation triggers the engulfment and degradation of mitochondria by autolysosomes, releasing mitochondrial DNA. The free mitochondrial DNA activates Toll-like receptor 9 (TLR9), which relocates from the ER to the lysosome, mediating the enrichment of the phosphatase OCRL. OCRL is induced by the downstream effector TRPML1 (a calcium channel crucial for autophagosome fusion) and AP2-recruited phosphokinases. The clearance of PtdIns(4,5)P2 by OCRL releases MCOLN1 from its inhibitory state, and the subsequent release of Ca2+ may form a positive feedback loop that promotes fusion [114], [115].

The existing literature on the spatiotemporal regulation of fusion by ions is limited and currently concentrates on TRPML1 and TRPML3. It is hypothesized that the spatiotemporal regulation of autophagy by ions may contain three terms. The initial consideration is the localization of ion channels. For instance, ion channels situated at the ER-mito contact sites have been demonstrated to influence the formation of omegasomes. Furthermore, the maturation and fusion of autophagosomes are predominantly regulated by endolysosomal and autophagosomal ion channels. To date, TRPML3 is the only ion channel that has been found to traffick onto the autophagic structure. Upon the induction of autophagy, C-terminal of TRPML3 is palmitoylated, resulting in the targeted transport of TRPML3 to the phagosome and its recruitment by the ATG8 family protein Golgi-associated ATPase enhancer of 16 kDa (GATE16), which promotes phagosome maturation [116], [117]. Interestingly, the three tandem histidine constitutes the pH sensor of TRPML3 [3], which may be stimulated by H+ to dissociate from the autophagosome during its proximity to the lysosome [118]. Given that GATE16 itself exhibits tethering activity, it can be hypothesized that TRPML3 inhibits the tethering activity of GATE16 [119], [120]. This inhibition occurs only when the autophagosome matures, at which point TRPML3 senses the increase in H+ and dissociates from GATE16, thus allowing fusion. The second point concerns the regulation of ion channels by phosphatidylinositol. TRPML1 activity is stimulated by PtdIns(3,5)P2 and inhibited by PtdIns(4,5)P2 [115], [121]. TRPML3 localization to phagosomes is also followed by specific binding to PtdIns3P (although not directly regulating TRPML3 activity) to promote phagosome maturation [118]. Thirdly, the extent of ion channel activation matters during membrane fusion, which has been comprehensively reviewed in another article [122].

We highlight the molecular basis of the organized autophagy process: effector molecules depend on the spatial and temporal regulation of phosphatidylinositols, Rab GTPases, and ATG8s. Through microtubule-dependent transport, the cell concentrates autophagosomes into a “processing center” (perinuclear region), which not only improves fusion efficiency and rate of delivery, but also facilitates signal integration and transcriptional regulation. At the same time, the synergy between effector and regulatory molecules provides the necessary mechanical energy and precise molecular docking for this process. This mechanism provides important clues to our understanding of the relationship between autophagy and a variety of diseases (e.g., neurodegenerative diseases, cancer, etc.), and also points the way to the future development of therapeutic strategies to regulate autophagy.

Autophagy fusion and diseases

In recent years, significant progress has been made in understanding the molecular mechanisms of autophagy, the regulation of autophagy, and the effects of autophagy on physiology and pathophysiology. autophagosome-lysosome fusion defects are mainly discussed in cancer or neurodegenerative diseases and have been elucidated in detail in previous reviews [1], [123]. We have therefore summarized the molecules and compounds that regulate autophagosome-lysosome fusion (Tables 1 and 2). Unfortunately, the clinical application of autophagy is still in its infancy. Among the autophagy inhibitors identified so far, only chloroquine (CQ) and hydroxychloroquine (HCQ) (both inhibit autophagosome-lysosome fusion) are approved for clinical use and are focused on cancer. The combination of CQ or HCQ with chemotherapeutic agents in patients with BRAF/MEK-resistant melanoma [124] or glioblastoma with epidermal growth factor receptor vIII mutations [125] has yielded relatively promising results, but has not significantly improved overall survival in most clinical trials [126], [127], [128], [129], [130]. There are a number of reasons why CQ / HCQ is not widely used in the clinical treatment of anti-tumors, such as the fact that the higher doses of HCQ required for therapeutic efficacy (>600 mg/day) often result in unacceptable adverse effects in clinical trials, making the dosing regimen clinically unfeasible [131]. In addition, although CQ and HCQ possess anti-cancer mechanisms related to autophagy, they also possess other mechanisms unrelated to autophagy, such as induction of apoptosis, necrotic apoptosis and immunomodulatory effects. The complete link between these different mechanisms, clinical efficacy and safety has not yet been fully established [132], [133]. In addition, kidney and intestinal tissues of HCQ-treated mice showed disorganization of the Golgi apparatus [134].

Table 1:

Localization and function of phosphatidylinositol.

Inositol phosphate Localization Function Reference
PtdIns4P converted to PtdIns(4,5)P2 Endosome PtdIns4P on endosomes is rapidly converted to PtdIns(4,5)P2, resulting in the dissociation of PLEKHM1 from Rab7 [86]
PtdIns(3,5)P2 converted to PtdIns3P Lysosome INPP5E is transported to the lysosome, where it catalyzes the conversion of PtdIns(3,5)P2 to PtdIns3P [102]
PtdIns4P Autophagosome PtdIns4P rapidly enriches and attracts free STX17 recruitment to autophagosome membranes after autophagosome closure [110]
PtdIns(4,5)P2 Autophagosome SNAP47’s positively charged amino acids are then drawn to the negatively charged PtdIns(4,5)P2 localized on the phagosome, leading to SNAP47’s recruitment to the phagosome. [145]

  1. STX, syntaxin.

Table 2:

Compounds targeting autophagosome-lysosome fusion.

Compounds Disease Mechanism Reference
Chloroquine and hydroxychloroquine Cancer, immune diseases and neurodegenerative diseases CQ or HCQ alleviate disease progression by inhibiting autophagosome-lysosome fusion [134], [173], [174]
Bafilomycin A1 – Braf A1 blocks autophagosome-lysosome fusion by inhibiting the calcium pump SERCA
NEO214 Glioblastoma NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. [175]
Crizotinib Cardiotoxicity Crizotinib causes mitochondrial damage, myocardial injury, and left ventricular dysfunction in mice by blocking autophagosome-lysosome fusion [176]
G42 Zika virus and murine hepatitis virus G42 alkalised lysosomal pH and inhibited autophagosome-lysosome fusion. [177]
C10 Breast cancer C10 induces early autophagy and blocks late autophagosome-lysosome fusion to inhibit breast cancer proliferation [178]
Thapsigargin – Thapsigargin specifically blocks autophagosome-lysosome fusion [179]
Vacuolin-1 – Vacuolin-1 inhibits autophagy by impairing lysosomal maturation via PIKfyve inhibition [180]
Bisphenol A Fatty liver BPA exposure decreases the translocation of Stx17 to lysosome resulting in the autophagogome-lysosome fusion defect. [181]
Berbamine Cancer BBM induces up-regulation of BNIP3 and promotes interaction between SNAP29 and BNIP3. [153]
Ir-VPA Cervical cancer Ir-VPA promotes apoptosis by inhibiting autophagosome-lysosome fusion [182]
DAC and DAS Cervical cancer DAC and DAS inhibit autophagosome fusion and enhance Hela sensitivity to campothecin by impairing lysosomal function [183], [184]
MHY1485 Hepatocellular carcinoma MHY1485 enhances the sensitivity of HepG2 cells to doxorubicin by inhibiting the fusion of autophagosomes and lysosomes. [185]
Albendazole Parkinson’s disease Albendazole promotes the aggregation of lysosomes around the nuclear periphery through a JIP4-TRPML1-dependent mechanism, facilitating the fusion of autophagosomes and lysosomes, and enhancing the degradation of protein aggregates. [186]
WX8 Melanoma The WX8 family selectively kills autophagy-dependent cancer cells by inhibiting autophagy. [187]
Anhydrodebromoaplysiatoxin Cancer Anhydrodebromoaplysiatoxin inhibits cancer cell proliferation by inducing autophagy and suppressing the fusion of late autophagosomes. [188]
Atractylenolide I Cancer Atractylenolide I upregulates ATPase subunit ATP6V0D2 (ATPase H+ transporter V0 subunit d2) to promote the autophagic degradation of EPAS1, thereby increasing lysosomal function and facilitating the fusion of autophagosomes and lysosomes, and ultimately inhibiting tumor angiogenesis. [189]
Aloperine NSCLC Aloperine downregulates VPS4A to inhibit cancer progression by suppressing autophagosome-lysosome fusion in NSCLC [139]
Andrographolide Cancer Andrographolide enhances cisplatin-induced apoptosis by inhibiting autophagosome-lysosome fusion in human cancer cells [190]

  1. CQ, chloroquine; HCQ, hydroxychloroquine; Braf A1, Bafilomycin A1; BPA, Bisphenol A; BBM, Berbamine.

The biggest difficulty in autophagy drug development is that we cannot determine whether the targeted autophagy molecules have autophagy-independent functions. Examples include the BECN1-PIK3C3/VPS34 complex, proteins that have autophagy-independent functions in other cellular processes [135]. Therefore, further exploration of autophagy mechanisms is crucial for the development of effective autophagy inhibitors.

Senescence is a biological process characterized by the decline of cells and functions over time, leading to a decrease in the quality of life of an organism. Studies in Caenorhabditis elegans and human cells have shown that autophagy declines with age, exacerbating cellular damage and leading to the development of age-related diseases [136], [137], [138]. Therefore, therapeutic strategies to enhance autophagy are often adopted for aging and age-related pathologies such as neurodegenerative diseases. Overexpression of Rab2, Arl8 or TECPR1 delays neuronal aging or ameliorates neurodegeneration [139], [140]. Metformin inhibits vascular smooth muscle cell senescence by promoting autophagosome-lysosome fusion [141]. Dehydroepiandrosterone prevents linoleic acid-induced endothelial cell senescence by increasing autophagy [142]. Olanzapine is one of the most commonly used antipsychotic drugs (APDs) for the treatment of psychiatric patients, but long-term exposure to APDs may lead to accelerated aging and cognitive decline. The main cause of this is impaired autophagosome-lysosome fusion, which results in the inability to clear damaged mitochondria. Olanzapine-induced mitochondrial fragmentation, shortened lifespan and poor health can be ameliorated by the mitophagy inducer urolithin A [143].

Summary

Autophagosome-lysosome fusion is one of the key rate-limiting steps in the late stages of autophagy. Recent research has identified several key molecules involved in the fusion process, including effector and regulatory molecules that facilitate this process. Nevertheless, numerous questions remain unanswered, including:

  1. The assembly and regulatory mechanisms of tetrameric t-SNARE complexes and hexametric HOPS complexes remain to be elucidated. In particular, the molecules involved in the recruitment, polymerization, assembly and depolymerization of SNAREs are yet to be identified. What is the initiating signal that triggers assembly of HOPS into dimers in the cytoplasm?

  2. Phosphatidylinositols, Rab GTPases, and ATG8s play pivotal roles in the spatiotemporal regulation of the fusion process. However, their dynamics (of activity, expression, translocation or mode of modification) during autophagy have rarely been studied.

  3. It remains unclear what is the energy source for fusion. Also, how the energy barrier is reduced in autophagosome-lysosome membrane fusion?

SNARE complexes and bolus proteins may utilize their own structural peculiarities to subtly generate mechanical energy and thus reduce the energy barrier. During autophagosome-lysosome fusion, one of the most energy-consuming steps is the depolymerization of SNAREs helical complexes. NSF (a molecular motor belonging to the AAA+ ATPase family) utilizes the chemical energy generated by the hydrolysis of ATP to convert it into mechanical energy, and applies torsional or tangential forces through a series of conformational changes to depolymerize SNARE four-helix bundles into individual SNARE proteins, thereby breaking the stable interactions within the complex. “Untwisting” or depolymerizing SNARE four-helix bundles into individual SNARE proteins through a series of conformational changes by applying torsional or tangential forces, thereby breaking the stabilizing interactions within the complex. The depolymerization of each SNARE complex may require the consumption of about 2–3 ATP molecules. V-ATPases themselves are proton pumps on the surface of lysosomes and maintain their acidity by feeding H+ to the lysosome. Thus, V-ATPases themselves do not produce energy, but they provide the basis for lysosomes to break down autophagic substrates by maintaining an acidic environment. Lysosomes degrade autophagic substrates, such as lipid droplets, sugars, or proteins, and produce them as free fatty acids, glucose, or amino acids, etc., which may enter into the mitochondria to produce energy and further sustain autophagy.

  1. It remains unclear which molecules sense autophagosome closure and send out the fusion signals. Exploring molecules such as ions or phosphatidylinositol that are perception-sensitive or easily converted may help to further the understanding of the problem.

  2. It remains unclear how the cytoskeleton controls the movement of autophagosomes toward lysosomes. The cytoskeleton plays a role mainly in the preparatory phase of autophagosome-lysosome fusion, i.e., allowing autophagosomes localized in the organelle and lysosomes localized in the periphery to transit to the vicinity of the perinuclear perimeter for confluence. Some of the molecules involved in this process are known, but how the cell senses when autophagosomes and lysosomes are required to move to the perinuclear area needs to be further explored.

  3. The exact role of ions in autophagosome-lysosome fusion.

  4. It is notable that in different tissues or upon different autophagy stimuli, there may be specific regulatory molecules that have rarely been studied. these specific molecules may provide potential organ-specific or precise disease-related autophagy-regulation target.

  5. The deletion or mutation of genes encoding effector or regulatory molecules during autophagosome-lysosome fusion is closely associated with neurodegenerative diseases. A comprehensive investigation of the fusion mechanism may facilitate the identification of therapeutic targets for these diseases.

Autophagy occurs in almost all human cells, and defects in autophagosome-lysosome fusion are frequently linked to developmental defects, infections, cancer, or neurodegenerative diseases [123], [144]. Further studies are required to elucidate the precise mechanism underlying autophagosome-lysosome fusion and to identify potential therapeutic targets for conditions associated with dysregulated autophagosome-lysosome fusion.

Corresponding author: Jiajie Diao, Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, 45267, USA, E-mail: ; and Shen Zhang and Qing Zhong, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200020, China, E-mail: (S. Zhang), (Q. Zhong)
Shizuo Liu and Huan Yan contributed equally to this work.

  1. Research ethics: Not applicable.

  2. Informed consent: Not applicable.

  3. Author contributions: S.L. and H.Y. mainly wrote the manuscript and drew the figures. S.Z., J.D. and Q.Z. revised the manuscript.

  4. Use of Large Language Models, AI and Machine Learning Tools: None declared.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: The work was supported in part by grants 92254307 (NSFC (National Natural Science Foundation of China)) to Q.Z., 2023YFA0914900 (MOST (Ministry of Science and Technology of the People’s Republic of China)) to Q.Z., 32361163613 (NSFC) to Q.Z., M-0140 (NSFC) to Q.Z., and innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20212000). The work was also supported by Shanghai Frontiers Science Center of Cellular Homeostasis and Human Diseases, and the Fundamental Research Funds for the Central Universities to Q.Z. lab.

  7. Data availability: Not applicable.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/mr-2024-0095).

Received: 2024-12-10
Accepted: 2025-05-20
Published Online: 2025-06-19

© 2025 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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https://doi.org/10.1515/mr-2024-0095
Keywords for this article
autophagosome-lysosome fusion; sensitive factor attachment protein receptor; syntaxin 17; autophagy
Creative Commons
BY-NC-ND 4.0

Articles in the same Issue

  1. Frontmatter
  2. Reviews
  3. The potential of generative AI with prostate-specific membrane antigen (PSMA) PET/CT: challenges and future directions
  4. Target discovery-directed pharmacological mechanism elucidation of bioactive natural products
  5. Spatio-temporal processes in autophagosome-lysosome fusion
  6. A review of 3D bioprinting for organoids
  7. Perspectives
  8. Can chimeric antigen receptors – based therapy bring a gleam of hope for thyroid-associated ophthalmopathy and other autoimmune diseases?
  9. Artificial intelligence-powered innovations in radiotherapy: boosting efficiency and efficacy
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