Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation inDrosophila

Abstract

complex required for transport; E(spl)mb-HLH, enhancer of split mb, helix-loop-helix; FE, follicular epithelium; histone H3, His3; hop-Stat92E, hopscotch-signal transducer and activator of transcription protein at 92E; GFP, green fluorescent protein; MENE, mutant eye no eclosion; MVB, multivesicular body; N, Notch; NECD, N extracellular domain; NPF, asparagine-proline-phenylalanine; os, outstretched; ref(2)P, refractory to sigma P; Snap29, synaptosomal-associated protein 29 kDa; SNARE, soluble NSF attachment protein receptor; Socs36E, suppressor of cytokine signaling at 36E; Syb, Synaptobrevin; Syx, syntaxin; Vamp, vesicle-associated membrane protein; V-ATPase, vacuolar H C -ATPase; Vps25, vacuolar protein sorting 25; WT, wild type.How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we find evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29.

Dedicated SNARE s and specialized TRIM cargo receptors mediate secretory autophagy

“…Incidentally, SNAP‐29 plays a role in autophagosome–lysosome fusion (Itakura et al , ; Hamasaki et al , ; Takats et al , ; Diao et al , ). However, it is also known to interact with plasma membrane syntaxins (Steegmaier et al , ) and thus far it has been noted for its regulatory, and even inhibitory roles (Su et al , ; Morelli et al , ). However, in most cases, it plays a role in fusion events (Xu et al , ).…”

Enteroviruses Remodel Autophagic Trafficking through Regulation of Host SNARE Proteins to Promote Virus Replication and Cell Exit

“…STX17 is the Qa SNARE on the autophagosome that coordinates its fusion with other vesicles (Itakura et al, 2012). SNAP29 is a cytosolic Qbc SNARE that binds to STX17, donating its two helices to the forming fusion bundle (Morelli et al, 2014). VAMP8 is an R SNARE, found on lysosomal and endosomal membranes, that binds to STX17-SNAP29, tethering the membranes together for fusion (Diao et al, 2015).…”

An essential step of kinetochore formation controlled by the SNARE protein Snap29

“…Compared to wild‐type disks, Snap29 mutant disks also present epithelial architecture defects characterized by appearance of mesenchymal‐like F‐actin‐rich cells (Appendix Fig S7A and B, arrowheads) (Morelli et al , ). Disks that are trans‐heterozygous for null mutations in Syx17 or Vamp7 , which act with Snap29 in autophagy, do not display epithelial alteration (Morelli et al , ) and localize Snap29 at KTs (Appendix Fig S7E and F), ruling out their involvement in Snap29 localization at KTs and a function of autophagy in tissue formation. In contrast, altered epithelial architecture is a reported consequence of aberrant cell division in Drosophila imaginal disks.…”

“…To determine how SNAP29 supports KT formation, we measured KNL1 localization to KTs in SNAP29 KD cells overexpressing siRNAresistant SNAP29 mutant forms. We observed that a full-length human SNAP29, a functional form of Drosophila Snap29 (Morelli et al, 2014), or a human SNAP29 mutant form lacking the first SNARE domain (SNARE1), all rescue KNL1 localization to KTs in SNAP29 KD cells, while a mutant lacking the second SNARE domain (SNARE2) is less efficient (Fig 6A and B; quantification in D; Appendix Fig S6A and B). Thus, KNL1 Snap29 ability to recruit KNL1 to KTs is conserved and requires the C-terminal SNARE domain.…”