Review www.cell-stress.com How to use a multipurpose SNARE: The emerging role of Snap29 in cellular health Valeria Mastrodonato1,#, Elena Morelli1,# and Thomas Vaccari1,* 1 Dipartimento di Bioscienze, Universita’ degli Studi di Milano, Italy. # Equal contribution. * Corresponding Author: Thomas Vaccari, Dipartimento di Bioscienze, Universita’ degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy; Tel: +39 02503- 14886 ; Fax: +39 02503-14880; E-mail: [email protected] ABSTRACT Despite extensive study, regulation of membrane trafficking is doi: 10.15698/cst2018.04.130 incompletely understood. In particular, the specific role of SNARE (Soluble Received originally: 09.02.2018 NSF Attachment REceptor) proteins for distinct trafficking steps and their in revised form: 08.03.2018, Accepted 08.03.2018, mechanism of action, beyond the core function in membrane fusion, are still Published 22.03.2018. elusive. Snap29 is a SNARE protein related to Snap25 that gathered a lot of attention in recent years. Here, we review the study of Snap29 and its emerg- ing involvement in autophagy, a self eating process that is key to cell adapta- Keywords: Snap29, membrane tion to changing environments, and in other trafficking pathways. We also trafficking, endocytosis, autophagy, SNARE proteins, SNAP family, cell discuss Snap29 role in synaptic transmission and in cell division, which might division. extend the repertoire of SNARE-mediated functions. Finally, we present evi- dence connecting Snap29 to human disease, highlighting the importance of Snap29 function in tissue development and homeostasis. Abbreviations: CEDNIK – cerebral dysgenesis, neuropathy, ichtyosis and keratoderma, HOPS – homotypic fusion and protein sorting, HPIV – human para influenza virus, IL – Interleukin, SNAP – synaptosomal-associated protein, SNARE - soluble N-ethylmaleimide- sensitive-factor attachment receptor, Syx – syntaxin. INTRODUCTION 4-helix bundle (often referred to as trans-SNARE complex) The specialized trafficking routes that evolved between composed of distinct SNARE domains named Qa-, Qb-, Qc- compartments of the endomembrane system rely on a or R-SNARE are invariantly required for fusion. Usually, a wide set of proteins, including primarily SNAREs, Rab Qa-SNARE-containing protein [often called syntaxin, or GTPases and a large set of effectors and tethering factors, target (t)-SNARE] and a R-SNARE -containing protein [often that ensure specificity of cargo delivery to a wide range of called vesicle associated membrane protein (VAMP) pro- different target compartments. Precision and fidelity of tein, or v-SNARE] are carried by opposing membranes, targeting is paramount to support cell viability and to pre- each providing a SNARE domain to the fusion complex. vent disease. However, how such specificity is achieved at These proteins are glued together by Qb- and Qc-SNARE a molecular level is incompletely understood. domain containing proteins, providing the remaining 2 SNAREs are part of the conserved coiled-coil machinery domains (Fig. 1A). After fusion, all proteins of the 4-helix that brings membranes in close proximity, a prerequisite bundle are associated to the target membrane, in an ar- for most membrane fusion events occurring during traffick- rangement often called cis-SNARE complex, that is disas- ing [1-3]. A stereotypic set of SNARE proteins forming a sembled by the activity of a number of proteins including OPEN ACCESS | www.cell-stress.com 72 Cell Stress | APRIL 2018 | Vol. 2 No. 4 V. Mastrodonato et al. (2018) Snap29 in cellular health FIGURE 1: Illustration of the trans-SNARE complex involved in membrane fusion. All SNARE domains are oriented with the N-termini to the left (A). Schematic representation of SNAP family members in mammals (B). The homologous SNARE domains among the 4 paralogs are shown. The position of the central glutamine residue (Q) is indicated by a line. The linker region of Snap29 is (purple) differs from that of other family members and does not contain a cysteine rich region (CRR; light blue) for membrane targeting.The N-Terminal acidic NPF motif, exclusive to Snap29 among family members, is shown in yellow. the ATPase NSF (N-ethylmaleimide-sensitive factor). The initial studies, when compared to the advanced under- Qb- and Qc-SNAREs domains can also be contributed in a standing of Snap25 and Snap23 function, the specificity single protein, as is the case of members of the Synapto- and molecular regulation of Snap29 function remained to somal-Associated Protein (SNAP) protein family. Snap25 be explored. Below, we review the growing body of evi- and Snap23 are the most extensively studied SNAP family dence indicating that Snap29 regulates membrane fusion members (for review [4]). Metazoan genomes further in- at multiple cellular locales during intracellular trafficking. In clude Snap29, while the SNAP family in mammals compris- addition, we report findings that indicate that Snap29 es also Snap47 [5, 6]. Here, we will focus on Snap29, whose might not only be involved in membrane fusion, but that it functions have increasingly come into view in the last two could rather serve a regulatory or structural role in certain decades. contexts. Finally, we discuss the involvement of Snap29 in Differently from other family members Snap29 possess human disease. an N-terminal acidic asparagine-proline-phenylalanine (NPF) motif. Similarly to Snap47, Snap29 also lacks cysteine ENDOCYTOSIS, RECYCLING AND CILIUM FORMATION residues in the spacer region between the two SNARE do- REQUIRE SNAP29 AND ITS INTERACTOR EHD1 mains, that in Snap25 and Snap23 are modified to allow Together with the NPF interactor EHD1 and with the endo- membrane anchoring [5] (Fig. 1B). Consistent with the lack cytic adapters AP2, Snap29 was reported to promote en- of a membrane anchor, early biochemical studies revealed dosomal trafficking of the Insulin Growth Factor 1 receptor that Snap29 is only partially associated to membranes [5]. (IGF-1R) in CHO cells, a process crucial to down-regulate They also suggested that Snap29 interacts with a wide active receptors [9] (Fig. 2A). range of syntaxins including Syntaxin6 (Syx6) a SNARE pro- Biochemical analyses showed that binding of EHD1 to tein acting in the Golgi apparatus [5, 7, 8]. Despite these the NPF motif of Snap29 can occur alternatively to that of the F-BAR protein syndapin II. Upon overexpression in HeLa cells, alternative EHD1 and syndapin II interaction to Snap29 affected internalization of transferrin receptors HIGHLIGHTS (TFR) but not of EGFR, suggesting that Snap29 might con- trol trafficking of selected receptors [10] (Fig. 2B). • Snap29 regulates endocytosis and recycling by More recently, Snap29 has been shown to participate, interacting with EHD1. both in RPE cells and in Danio rerio, in trafficking of ciliary vesicles, a process that depends on EHD1, on the recycling • Snap29 is a key regulator of autophagy and assists GTPase Rab11, and on the Golgi apparatus GTPase Rab8 specialized secretion. [11]. The authors suggest that EHD1 is required for vesicle • Snap29 non canonical activities include regulation formation from preexisting membranes that fuse together and assemble in a membrane structure enveloping the of cell division and synaptic transmission. cilium microtubules (MTs), by the action of Snap29- • Alteration of Snap29 is observed in congenital, containing SNARE complexes (Fig. 2C). Interaction of neurological and infectious disease. Snap29 with EHD1 was also reported in Drosophila mela- nogaster [12]. OPEN ACCESS | www.cell-stress.com 73 Cell Stress | APRIL 2018 | Vol. 2 No. 4 V. Mastrodonato et al. (2018) Snap29 in cellular health by self-digesting cytoplasmic molecules. Autophagic clear- ance requires the formation of an intermediate double- membrane organelle called autophagosome that fully en- gulfs the cargo before fusion with lysosome (for review [13]). In 2012, Itakura et al. reported that fusion of starva- tion-induced autophagosomes with lysosomes in HeLa cells requires Syntaxin17 (Syx17), Snap29 and the lysosomal R- SNARE Vamp8 [14]. In such work, the authors showed that Syx17 is recruited to autophagosomes from the endo- plasmic reticulum (ER) and that Snap29 primarily associate to it, possibly from the cytoplasm. Subsequent characteri- zation of Syx17 and Snap29 in D. melanogaster revealed a similar role in autophagy in vivo [15, 16]. The mechanism of Snap29-dependent regulation of au- tophagy was further dissected in human cells by showing that the binary Syx17-Snap29 complex on autophagosomes is stabilized by binding of ATG14 oligomers to Syx17. Such tethering function is proposed to be crucial for priming the complex for fusion with Vamp8 on the lysosomes [17]. Interestingly, analysis of Caenorhabditis elegans mu- tants indicated that O-GlcNac modification of Snap29 is crucial to regulate autophagic clearance [18]. In fact, in FIGURE 2: Roles of Snap29 in endocytosis and recycling. both worms and in human cells, it was found that in pres- Snap29 is required for internalization of the IGF-1R receptor ence of high nutrients Snap29 is modified on 4 Serine resi- (A), for internalization and/or recycling of TFR (B) and for for- dues, thus preventing inclusion in a functional SNARE com- mation of the basal membrane surrounding the cilium microtu- plex. In contrast, in low nutrient conditions such as starva- bules (light blue; C). All these functions are regulated in associa- tion with the endocytic factor EHD1, which binds the NPF motif tion, the unmodified Snap29 is