Mediatory molecules that fuse autophagosomes and lysosomes

Autophagy functions to degrade intracellular foreign microbial invaders by a process that is termed xenophagy (antimicrobial autophagy). Xenophagosomes undergo a stepwise maturation process culminating in a fusion event with lysosomes, after which the cargos are degraded. Recent investigations by our laboratory demonstrate that endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are involved in the fusion between xenophagosomes and lysosomes. Knockdown of the combinational SNARE proteins Vti1b and VAMP8 with siRNAs disturbs the colocalization of LC3 with LAMP-1. We also find that the invasive efficiency of group A Streptococcus into cells is not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency is significantly diminished, indicating that xenophagy is functionally impaired. In addition, knockdown of these SNAREs inhibits the fusion of canonical autophagosomes with lysosomes. Together, these findings indicate that VAMP8 and Vti1b mediate fusion with lysosomes in both antimicrobial and canonical autophagy.     

Combinational Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Proteins VAMP8 and Vti1b Mediate Fusion of Antimicrobial and Canonical Autophagosomes with Lysosomes

Autophagy plays a crucial role in host defense, termed antimicrobial autophagy (xenophagy), as it functions to degrade intracellular foreign microbial invaders such as group A Streptococcus (GAS). Xenophagosomes undergo a stepwise maturation process consisting of a fusion event with lysosomes, after which the cargoes are degraded. However, the molecular mechanism underlying xenophagosome/lysosome fusion remains unclear. We examined the involvement of endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in xenophagosome/lysosome fusion. Confocal microscopic analysis showed that SNAREs, including vesicle-associated membrane protein (VAMP)7, VAMP8, and vesicle transport through interaction with t-SNAREs homologue 1B (Vti1b), colocalized with green fluorescent protein-LC3 in xenophagosomes. Knockdown of Vti1b and VAMP8 with small interfering RNAs disturbed the colocalization of LC3 with lysosomal membrane protein (LAMP)1. The invasive efficiency of GAS into cells was not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency was significantly diminished, indicating that antimicrobial autophagy was functionally impaired. Knockdown of Vti1b and VAMP8 also disturbed colocalization of LC3 with LAMP1 in canonical autophagy, in which LC3-II proteins were negligibly degraded. In contrast, knockdown of Syntaxin 7 and Syntaxin 8 showed little effect on the autophagic fusion event. These findings strongly suggest that the combinational SNARE proteins VAMP8 and Vti1b mediate the fusion of antimicrobial and canonical autophagosomes with lysosomes, an essential event for autophagic degradation.     

Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events

Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans-SNARE complex consisting of Q-SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R-SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell-free systems, we confirmed this result, but found that the same Q-SNAREs can combine with an alternative R-SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co-immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N-terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.     

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L

Coxiella burnetii is a gram‐negative intracellular bacterium that forms a large, lysosome‐like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol‐binding protein‐related protein 1 long (ORP1L) is a mammalian lipid‐binding protein that plays a dual role in cholesterol‐dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N‐terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co‐localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L‐depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.     

Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development

Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N‐ethylmaleimide‐sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI‐VAMP/VAMP7 and cellubrevin/VAMP3, two v‐SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI‐VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development.     

Identification of OmpA,a Coxiella burnetii Protein Involved in Host Cell Invasion,by Multi-Phenotypic High-Content Screening

Coxiella burnetii is the agent of the emerging zoonosis Q fever. This pathogen invades phagocytic and non-phagocytic cells and uses a Dot/Icm secretion system to co-opt the endocytic pathway for the biogenesis of an acidic parasitophorous vacuole where Coxiella replicates in large numbers. The study of the cell biology of Coxiella infections has been severely hampered by the obligate intracellular nature of this microbe, and Coxiella factors involved in host/pathogen interactions remain to date largely uncharacterized. Here we focus on the large-scale identification of Coxiella virulence determinants using transposon mutagenesis coupled to high-content multi-phenotypic screening. We have isolated over 3000 Coxiella mutants, 1082 of which have been sequenced, annotated and screened. We have identified bacterial factors that regulate key steps of Coxiella infections: 1) internalization within host cells, 2) vacuole biogenesis/intracellular replication, and 3) protection of infected cells from apoptosis. Among these, we have investigated the role of Dot/Icm core proteins, determined the role of candidate Coxiella Dot/Icm substrates previously identified in silico and identified additional factors that play a relevant role in Coxiella pathogenesis. Importantly, we have identified CBU_1260 (OmpA) as the first Coxiella invasin. Mutations in ompA strongly decreased Coxiella internalization and replication within host cells; OmpA-coated beads adhered to and were internalized by non-phagocytic cells and the ectopic expression of OmpA in E. coli triggered its internalization within cells. Importantly, Coxiella internalization was efficiently inhibited by pretreating host cells with purified OmpA or by incubating Coxiella with a specific anti-OmpA antibody prior to host cell infection, suggesting the presence of a cognate receptor at the surface of host cells. In summary, we have developed multi-phenotypic assays for the study of host/pathogen interactions. By applying our methods to Coxiella burnetii, we have identified the first Coxiella protein involved in host cell invasion.     

From neglected to dissected: How technological advances are leading the way to the study of Coxiella burnetii pathogenesis

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for severe worldwide outbreaks of the zoonosis Q fever. The remarkable resistance to environmental stress, extremely low infectious dose and ease of dissemination, contributed to the classification of C. burnetii as a class B biothreat. Unique among intracellular pathogens, C. burnetii escapes immune surveillance and replicates within large autophagolysosome‐like compartments called Coxiella‐containing vacuoles (CCVs). The biogenesis of these compartments depends on the subversion of several host signalling pathways. For years, the obligate intracellular nature of C. burnetii imposed significant experimental obstacles to the study of its pathogenic traits. With the development of an axenic culture medium in 2009, C. burnetii became genetically tractable, thus allowing the implementation of mutagenesis tools and screening approaches to identify its virulence determinants and investigate its complex interaction with host cells. Here, we review the key advances that have contributed to our knowledge of C. burnetii pathogenesis, leading to the rise of this once‐neglected pathogen to an exceptional organism to study the intravacuolar lifestyle.     

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

Coxiella burnetii is an intracellular bacterial pathogen which causes Q fever, a human infection with the ability to cause chronic disease with potentially life-threatening outcomes. In humans, Coxiella infects alveolar macrophages where it replicates to high numbers in a unique, pathogen-directed lysosome-derived vacuole. This compartment, termed the Coxiella-containing vacuole (CCV), has a low internal pH and contains markers both of lysosomes and autophagosomes. The CCV membrane is also enriched with CLTC (clathrin heavy chain) and this contributes to the success of the CCV. Here, we describe a role for CLTC, a scaffolding protein of clathrin-coated vesicles, in facilitating the fusion of autophagosomes with the CCV. During gene silencing of CLTC, CCVs are unable to fuse with each other, a phenotype also seen when silencing genes involved in macroautophagy/autophagy. MAP1LC3B/LC3B, which is normally observed inside the CCV, is excluded from CCVs in the absence of CLTC. Additionally, this study demonstrates that autophagosome fusion contributes to CCV size as cell starvation and subsequent autophagy induction leads to further CCV expansion. This is CLTC dependent, as the absence of CLTC renders autophagosomes no longer able to contribute to the expansion of the CCV. This investigation provides a functional link between CLTC and autophagy in the context of Coxiella infection and highlights the CCV as an important tool to explore the interactions between these vesicular trafficking pathways.     

The cytoplasmic domain of Vamp4 and Vamp5 is responsible for their correct subcellular targeting: the N-terminal extenSion of VAMP4 contains a dominant autonomous targeting signal for the trans-Golgi network

SNAREs represent a superfamily of proteins responsible for the last stage of docking and subsequent fusion in diverse intracellular membrane transport events. The Vamp subfamily of SNAREs contains 7 members (Vamp1, Vamp2, Vamp3/cellubrevin, Vamp4, Vamp5, Vamp7/Ti-Vamp, and Vamp8/endobrevin) that are distributed in various post-Golgi structures. Vamp4 and Vamp5 are distributed predominantly in the trans-Golgi network (TGN) and the plasma membrane, respectively. When C-terminally tagged with enhanced green fluorescent protein, the majority of Vamp4 and Vamp5 is correctly targeted to the TGN and plasma membrane, respectively. Swapping the N-terminal cytoplasmic region and the C-terminal membrane anchor domain between Vamp4 and Vamp5 demonstrates that the N-terminal cytoplasmic region of these two SNAREs contains the correct subcellular targeting information. As compared with Vamp5, Vamp4 contains an N-terminal extension of 51 residues. Appending this 51-residue N-terminal extension onto the N terminus of Vamp5 results in targeting of the chimeric protein to the TGN, suggesting that this N-terminal extension of Vamp4 contains a dominant and autonomous targeting signal for the TGN. Analysis of deletion mutants of this N-terminal region suggests that this TGN-targeting signal is encompassed within a smaller region consisting of a di-Leu motif followed by two acidic clusters. The essential role of the di-Leu motif and the second acidic cluster was then established by site-directed mutagenesis.     

LAMP proteins account for the maturation delay during the establishment of the Coxiella burnetii‐containing vacuole

The obligate intracellular pathogen Coxiella burnetii replicates in a large phagolysosomal‐like vacuole. Currently, both host and bacterial factors required for creating this replicative parasitophorous C. burnetii‐containing vacuole (PV) are poorly defined. Here, we assessed the contributions of the most abundant proteins of the lysosomal membrane, LAMP‐1 and LAMP‐2, to the establishment and maintenance of the PV. Whereas these proteins were not critical for uptake of C. burnetii, they influenced the intracellular replication of C. burnetii. In LAMP‐1/2 double‐deficient fibroblasts as well as in LAMP‐1/2 knock‐down cells, C. burnetii establishes a significantly smaller, yet faster maturing vacuole, which harboured more bacteria. The accelerated maturation of PVs in LAMP double‐deficient fibroblasts, which was partially or fully reversed by ectopic expression of LAMP‐1 or LAMP‐2, respectively, was characterized by an increased fusion rate with endosomes, lysosomes and bead‐containing phagosomes, but not by different fusion kinetics with autophagy vesicles. These findings establish that LAMP proteins are critical for the maturation delay of PVs. Unexpectedly, neither the creation of the spacious vacuole nor the delay in maturation was found to be prerequisites for the intracellular replication of C. burnetii.     

Syntaxin 11 binds Vti1b and regulates late endosome to lysosome fusion in macrophages

Syntaxin 11 (Stx11) is a SNARE protein enriched in cells of the immune system. Loss or mutation of Stx11 results in familial hemophagocytic lymphohistiocytosis type-4 (FHL-4), an autosomal recessive disorder of immune dysregulation characterized by high levels of inflammatory cytokines along with defects in T-cell and natural killer cell function. We show here Stx11 is located on endosomal membranes including late endosomes and lysosomes in macrophages. While Stx11 did not form a typical trans-SNARE complex, it did bind to the Q-SNARE Vti1b and was able to regulate the availability of Vti1b to form the Q-SNARE complexes Stx6/Stx7/Vtib and Stx7/Stx8/Vti1b. The mutant form of Stx11 sequestered Vti1b from forming the Q-SNARE complex that mediates late endosome to lysosome fusion. Depletion of Stx11 in activated macrophages leads to an accumulation of enlarged late endocytic compartments, increased trafficking to the cell surface and inhibition of late endosome to lysosome fusion. These phenotypes are rescued by the expression of an siRNA-resistant Stx11 construct in Stx11-depleted cells. Our results suggest that by regulating the availability of Vti1b, Stx11 regulates trafficking steps between late endosomes, lysosomes and the cell surface in macrophages.     

Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion

Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.     

Rab7 and Arl8 GTPases are Necessary for Lysosome Tubulation in Macrophages

Lysosomes provide a niche for molecular digestion and are a convergence point for endocytic trafficking, phagosome maturation and autophagy. Typically, lysosomes are small, globular organelles that appear punctate under the fluorescence microscope. However, activating agents like phorbol esters transform macrophage lysosomes into tubular lysosomes (TLs), which have been implicated in retention of pinocytic uptake and phagosome maturation. Moreover, dendritic cells exposed to lipopolysaccharides (LPSs) convert their punctate class II major histocompatibility complex compartment, a lysosome‐related organelle, into a tubular network that is thought to be involved in antigen presentation. Other than a requirement for microtubules and kinesin, little is known about the molecular mechanisms that drive lysosome tubulation. Here, we show that macrophage cell lines readily form TLs after LPS exposure, with a requirement for the Rab7 GTPase and its effectors RILP (Rab7‐interacting lysosomal protein) and FYCO1 (coiled‐coil domain‐containing protein 1), which respectively modulate the dynein and kinesin microtubule motor proteins. We also show that Arl8B, a recently identified lysosomal GTPase, and its effector SKIP, are also important for TL biogenesis. Finally, we reveal that TLs are significantly more motile than punctate lysosomes within the same LPS‐treated cells. Therefore, we identify the first molecular regulators of lysosome tubulation and we show that TLs represent a more dynamic lysosome population.     

Nuclear trafficking of the anti‐apoptotic Coxiella burnetii effector protein AnkG requires binding to p32 and Importin‐α1

The obligate intracellular bacterium Coxiella burnetii causes the zoonotic disease Q‐fever. Coxiella pathogenesis depends on a functional type IV secretion system (T4SS). The T4SS effector AnkG inhibits pathogen‐induced host cell apoptosis, which is believed to be important for the establishment of a persistent infection. However, the mode of action of AnkG is not fully understood. We have previously demonstrated that binding of AnkG to p32 is crucial for migration of AnkG into the nucleus and that nuclear localization of AnkG is essential for its anti‐apoptotic activity. Here, we compared the activity of AnkG from the C. burnetii strains Nine Mile and Dugway. Although there is only a single amino acid exchange at residue 11, we observed a difference in anti‐apoptotic activity and nuclear migration. Mutation of amino acid 11 to glutamic acid, threonine or valine results in AnkG mutants that had lost the anti‐apoptotic activity and the ability to migrate into the nucleus. We identified Importin‐α1 to bind to AnkG, but not to the mutants and concluded that binding of AnkG to p32 and Importin‐α1 is essential for migration into the nucleus. Also during Coxiella infection binding of AnkG to p32 and Importin‐α1 is crucial for nuclear localization of AnkG.     

Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion.

Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p.     

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes,is essential for the biogenesis of secretory organelles

Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome‐like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin‐like receptor and syntaxin‐6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab‐GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps‐C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal‐related compartments, the vacuole and immature apical secretory organelles. Conditional knock‐down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock‐down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite‐specific functions in the biogenesis of their unique secretory organelles.     

Autophagosomes: A Fast-Food Joint for Unexpected Guests

Coxiella burnetii is a Gram-negative obligate intracellular bacterium that infects a wide range of hosts including humans, causing Q fever, a disease characterized by high fever and flu-like symptoms. After its internalization the Coxiella-containing phagosomes interact with intracellular compartments and generate a large replicative vacuole that displays certain characteristics of a phagolysosome. We have shown that this bacterially-customized replicative vacuole also has the hallmarks of an autophagosomal compartment. Furthermore, in a recent publication we have reported that induction of autophagy is beneficial for the replication and survival of Coxiella. Different morphological forms of this bacterium have been described during its developmental cycle. Here we present additional data and discuss a model indicating that induction of autophagy favors the differentiation of the Coxiella small cell variants to the metabolically active large cells variants. We postulate that nutrient acquisition, likely by fusion with the nutrient-rich autophagic vacuoles, triggers the development of the LCVs, which actively multiply in the host cell.     

Molecular evidence for a novel <Emphasis Type="Italic">Coxiella</Emphasis> from <Emphasis Type="Italic">Argas monolakensis</Emphasis> (Acari: Argasidae) from Mono Lake,California, USA

Argasid ticks are vectors of viral and bacterial agents of humans and animals. Recent reports indicate that some ornithophilic argasids harbored rickettsial agents. A Nearctic tick, Argas monolakensis Schwan, Corwin, Brown is ornithophilic and has not previously been examined for rickettsial agents. Thirty adult A. monolakensis were tested by PCR for DNA from Rickettsia or Coxiella. Amplicons from a Coxiella sp. that were divergent from Coxiella burnetii were detected in 16/30 A. monolakensis. These molecular isolates were similar but not identical to C. burnetii, the Coxiella spp. of other ticks, and “Coxiella cheraxi” a pathogen of crayfish. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

The multi-functional SNARE protein Ykt6 in autophagosomal fusion processes

Autophagy is a degradative pathway in which cytosolic material is enwrapped within double membrane vesicles, so-called autophagosomes, and delivered to lytic organelles. SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins are key to drive membrane fusion of the autophagosome and the lytic organelles, called lysosomes in higher eukaryotes or vacuoles in plants and yeast. Therefore, the identification of functional SNARE complexes is central for understanding fusion processes and their regulation. The SNARE proteins Syntaxin 17, SNAP29 and Vamp7/VAMP8 are responsible for the fusion of autophagosomes with lysosomes in higher eukaryotes. Recent studies reported that the R-SNARE Ykt6 is an additional SNARE protein involved in autophagosome-lytic organelle fusion in yeast, Drosophila, and mammals. These current findings point to an evolutionarily conserved role of Ykt6 in autophagosome-related fusion events. Here, we briefly summarize the principal mechanisms of autophagosome-lytic organelle fusion, with a special focus on Ykt6 to highlight some intrinsic features of this unusual SNARE protein.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.