The SM Protein Car/Vps33A Regulates SNARE-mediated Trafficking to Lysosomes and Lysosome-related Organelles

Abstract

The SM proteins Vps33A and Vps33B are believed to act in membrane fusions in endosomal pathways, but their specific roles are controversial. In Drosophila, Vps33A is the product of the carnation (car) gene. We generated a null allele of car to test its requirement for trafficking to different organelles. Complete loss of car function is lethal during larval development. Eye-specific loss of Car causes late, light-independent degeneration of photoreceptor cells. Earlier in these cells, two distinct phenotypes were detected. In young adults, autophagosomes amassed indicating that their fusion with lysosomes requires Car. In eye discs, endocytosed receptors and ligands accumulate in Rab7-positive prelysosomal compartments. The requirement of Car for late endosome-to-lysosome fusion in imaginal discs is specific as early endosomes are unaffected. Furthermore, lysosomal delivery is not restored by expression of dVps33B. This specificity reflects the distinct pattern of binding to different Syntaxins in vitro: dVps33B predominantly binds the early endosomal Avl and Car to dSyntaxin16. Consistent with a role in Car-mediated fusion, dSyntaxin16 is not restricted to Golgi membranes but also present on lysosomes.

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

“…Perturbation of this delivery can be monitored using a weakly expressing chimeric reporter consisting of lumenal GFP fused to the combined transmembrane and cytosolic domain of human LAMP1 [28]. Upon transfer to lysosomes, hydrolases quickly degrade the lumenal GFP, thereby eliminating the GFP signal.…”

“…Fusion of the relatively neutral autophagosomes to acidic degradative LEs and lysosomes selectively quenches the more pH-sensitive and proteolytically labile GFP fluorescence, shifting the emitted fluorescence ratio from green/yellow towards red [52]. Autophagy is active in Drosophila photoreceptor cells, as evidenced by massive accumulation of autophagosomes in the eyes of flies with loss-of-function mutations in the HOPS complex components car/Vps33A and dor/Vps18 , due to a failure of HOPS-dependent autophagosome-lysosome fusion [28,49]. Consistent with this observation, expression of a GFP-mCherry-Atg8a transgene under an endogenous promoter produced strong red fluorescence in the developing ommatidia in wild-type L3 eye imaginal discs (Figure 2(f)).…”

“…prospero-Gal4 was a gift from Dr. Fumio Matsuzaki, RIKEN Center for Developmental Biology, Kobe, Japan. αTub84B-GFP-LAMP [28] was a gift from Dr. Helmut Krämer (University of Texas Southwestern Medical Center). The αTub84B-Rab7.YFP strain [92] was a gift from Dr. Suzanne Eaton (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany).…”

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

“…It is unclear, however, whether VPS33.1 and VPS33.2 are direct orthologous for mammalian VPS33A and VPS33B or reflect other evolutionary divergence. Similar to mammals, Drosophila VPS33A (Carnation) and VPS33B have distinct functions (38) and Drosophila VPS16 interacts with VPS33A, whereas Drosophila VIPAS39 interacts with VPS33B (39). However, Drosophila VIPAS39 also interacted with VPS18, suggesting that VIPAS39-VPS33B in Drosophila can interact with CORVET/ HOPS core subunits (39).…”

CORVET and HOPS tethering complexes–coordinators of endosome and lysosome fusion

“…Such a membrane binding would then replace the in vivo recruitment of HOPS by means of Ypt7-GTP, although at least one membrane-binding site is also crucial for HOPS function in vivo ) (see below). Once it is targeted to membranes, HOPS then catalyzes membrane fusion, presumably by capturing, chaperoning and proofreading SNAREs at the fusion site through its Vps33 subunit (Akbar et al, 2009;Krämer and Ungermann, 2011;Lobingier and Merz, 2012;Pieren et al, 2010;Starai et al, 2008) (Fig. 3A).…”