As mentioned above, SNAP29 is directly involved in autophagy through controlling the fusion of autophagosomal membrane with the lysosomal membrane (Itakura et al, 2012;
Takats et al, 2013) (see Chapter 1.6.2). Autophagy is an intracellular degradation process conserved among eukaryotes in which cytoplasmic contents are degraded within the lysosome. The resulting macromolecular constituents are recycled to generate energy to maintain cell viability (reviewed by (Feng et al, 2014)). There are three different types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy.
Macroautophagy is considered to be the prevalent form and is best characterized at the molecular level (hereafter referred to as autophagy; other types of autophagy are not discussed here) (reviewed by (Lamb et al, 2013)). It was originally believed that autophagy is a non-selective process (Kominami et al, 1983; Kopitz et al, 1990), induced as a survival mechanism in response to cellular stress. Over the past several years, however, a consensus has emerged that autophagosomal membranes can selectively recognize specific organelles and proteins, performing a cellular quality control. The most noted inducer of autophagy is nutrient starvation, but it can also be initiated by other stress stimuli, such as pharmacological agents (e.g. rapamycin and bafilomycin A1) (Barth et al, 2010) or endoplasmic reticulum
(ER) stress (Ogata et al, 2006). It is therefore useful to distinguish between basal and induced autophagy (Mizushima, 2005).
Autophagy is initiated by the formation of an isolation membrane, which sequesters a region of the cytoplasm, containing cytosolic proteins and organelles. Subsequently, this structure expands around the targeted material and is finally sealed, forming autophagosomes, which represent specialized double-membrane organelles. The formation of the pre-autophagosomal structures is mainly regulated by the function of autophagy-related (Atg) proteins, which operate in a strict hierarchical order (Itakura & Mizushima, 2010). Upon induction of autophagy a cytosolic form of microtubule-associated protein light chain 3 (LC3-I) is conjugated to phosphatidylethanolamine (PE) converting it into LC3-PE (LC3-II), which is associated with both the inner and outer autophagosomal membranes (reviewed by (Mizushima, 2007)). Autophagosomes transport their cargo to the lysosomes for degradation, where the outer membrane of the autophagosome fuses with the lysosome, converting it into an autolysosome (see Figure 7). Subsequently, the cytoplasmic content together with the inner membrane of the autphagosome are degraded by lysosomal hydrolases. Likewise, LC3-II is degraded in the autolysosomal lumen.
The origin of the isolation membrane remains unclear. However, it has become increasingly evident that in mammals it is originating from ER membranes (reviewed by (Lamb et al, 2013)). In addition, the expansion of the nascent autophagosome requires several other cellular compartments, such as the Golgi (van der Vaart & Reggiori, 2010), the plasma membrane (Ravikumar et al, 2010) and mitochondria (Hailey et al, 2010).
1.6.1 Measurement of autophagic flux
In order to monitor autophagic activity and to further distinguish between basal and induced autophagy, several assays have been established. One important aspect should be considered in this context: autophagic activity does not directly correspond to the accumulation of autophagosmomes. Therefore, monitoring the number of autophagosomes is insufficient to measure autophagic activity. An accumulation of autophagosomes might be due to an induction of autophagy (e.g. by nutrient starvation) or a block in downstream steps, respectively. Therefore, it is important to measure the “autophagic flux”, which enables to distinguish between these two possibilities. The term describes the dynamic process of autophagosome synthesis, the delivery of autophagosome cargoes to the lysosome, and the subsequent proteolytical degradation of these components (Mizushima & Levine, 2010).
Autophagic flux can be monitored by the turnover of the autophagosomal marker LC3-II (see figure 7), under the treatment with exogenous reagents like Bafilomycin A1, which prevents autophagosome-lysosome fusion and acts by inhibiting the vacuolar H+ ATPase (Yamamoto et al, 1998). As a result, autophagosomal structures accumulate (Klionsky et al, 2008;
Rubinsztein et al, 2009). However, measurement of LC3 turnover is not sufficient in order to monitor autophagic flux. Levels of other autophagy substrates, including p62 (also known as SQSTM1) have to be used to determine autophagic flux. p62 is incorporated into autophagosomes through direct binding to LC3 and it is subsequently degraded by the lysosome (Bjorkoy et al, 2005). Therefore, inhibition of autophagy leads to the accumulation of p62, whereas decreased levels of p62 are observed when autophagy is induced.
Figure 7: Incorporation of LC3-II and p62 into the autophagosome.
Cytosolic LC3-I is conjugated to PE and this step promotes integration of LC3-II into lipid membranes at the phagophore and autophagosomes. LC3B-II correlates with increased levels of autophagic vesicles. The ratio between LC3-I and LC3-II was used to determine changes in autophagosomal structures. The mammalian autophagy-specific substrate p62 interacts with poly-ubiquitinated protein aggregates and is incorporated into autophagosomes through direct binding to LC3-II. LC3-II as wells as p62 are subsequently degraded by the lysosome.
Accumulation of p62 indicates a block in autophagic flux. The illustration was kindly provided by Dr. Stina Schiller.
1.6.2 Involvement of SNAP29 in autophagy
Recently it has been shown in Drosophila melanogaster that Syntaxin17 (Stx17) is recruited to the outer membrane of autophagosomes to mediate fusion through its interactions with SNAP29 and VAMP7 (Takats et al, 2013). The fusion between autophagosome and lysosome is mediated by the three SNARE proteins: Stx17 (Qa-SNARE), SNAP29 (Qbc-SNARE), and the R-SNARE VAMP8 in human or VAMP7 in Drosophila melanogaster, respectively (see Figure 9) (Itakura et al, 2012; Takats et al, 2013). A knock-down of these genes in mammals resulted in accumulation of autophagosomes and a block of autolysosomal degradation during basal and starvation-induced autophagy. Furthermore, a role for Stx17 and its interaction partners in autophagy had also emerged in two other recent studies in mammals (Hamasaki et al, 2013; Itakura et al, 2012). Stx17 was found to be located on autophagosomes and knockdown of Stx17 resulted in a blockage of autophagic flux. The formation of autophagosomes was not blocked, but knockdown of Stx17 blocked the fusion of autophagosmes with lysosomes. Therefore, Stx17 is required for the fusion of autophagosomes with lysosomes in mammalian cells (Itakura et al, 2012).
In addition, a recent study reported that SNAP29 activity and, correspondingly, autophagy are regulated by post-translational addition of O-linked β-N-acetylglucosamine in Caenorhabditis elegans and HeLa cells (Guo et al, 2014). O-GlcNAcylation is a common post-translational modification that occurs on cytosolic as well as nuclear proteins (Mizushima, 2014). It has been established that the O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) modifies SNAP29 in a nutrient-dependent manner. This sugar modification of SNAP29 inhibits autophagosome-lysosome fusion as well as autophagic flux (Guo et al, 2014).
Furthermore, it is reduced under starvation conditions, suggesting that signals under nutrient-poor conditions can activate the fusion of autophagosomes with lysosomes by blocking O-GlcNAcylation of SNAP29 (see Figure 8) (Mizushima, 2014).
Figure 8: Regulation of autophagosome–lysosome fusion (modified from Mizushima 2014).
At the initiation step of autophagy the isolation membrane sequesters a part of the cytoplasm and eventually the autophagosome is formed. Afterwards, autophagosome-lysosome fusion proceeds in a process mediated by three SNARE proteins, Stx17 (blue), SNAP29 (green), and VAMP8 (orange). O-GlcNAcylation of SNAP29 (red symbols) by O-GlcNAc transferase (OGT) inhibits the formation of the SNARE complex, and thereby the fusion between the autophagosome and lysosome.