After infection with Salmonella those
After infection with Salmonella those bacteria that are not targeted by autophagy of the complete Salmonella-containing phagosome (see above) but are living freely in the cytosol can be eliminated by xenophagy, that is direct targeting of the ‘naked’ bacteria to the autophagic pathway. The type III secretion system of Salmonella contributes to release of the bacteria from the phagosome to the cytosol and this is followed by a rapid coating of the microbes by ubiquitin and association with autophagy receptors and LC3 (Microtubule-associated protein 1A/1B-light chain 3)-containing autophagosomes (Birmingham et al., 2006). The identity of the targeted bacterial components is unknown. Similarly, Mycobacterium tuberculosis can damage vacuoles by a bacterial type VII secretion system and provoke autophagy in macrophages. Following the damage, released bacteria are ubiquitinated by the ubiquitin ligase parkin followed by modification of the microorganisms with LC3 and adaptor proteins and wrapping into autophagosomes (Huang and Brumell, 2014).
MTORC1 integrates intra- and extracellular stimuli, and, when activated, promotes cell growth while inhibiting autophagy. High concentrations of amino acids inside the lysosome lead to recruitment of mTORC1 to the lysosome surface (Sancak et al., 2010). The molecular machinery for this process involves the Ragulator complex on the cytosolic face of the lysosome membrane and the associated RAG (Ras-related GTP-binding)-proteins. Once on the lysosome, mTORC1 is activated by the small lysosome-associated GTPase Rheb. Intraluminal arginine and glutamine concentrations are sensed by a recently identified amino PRIMA-1MET synthesis transporter (SLC38A9) working in cooperation with the V-ATPase complex (Jewell et al., 2015, Rebsamen et al., 2015, Zoncu et al., 2011). V-ATPase binds to the transporter through the subunits VOd1 and V1B2 (Wang et al., 2015). In addition, V-ATPase subunits interact with the lysosome-resident Ragulator complex and activate Ragulator when amino acid concentrations in lysosomes are high (Jewell et al., 2015, Wang et al., 2015, Zhang et al., 2014, Zoncu et al., 2011). Using this pathway, V-ATPase may upregulate autophagy when amino acid concentrations are low, for example during nutrient shortage.
The role of the V-ATPase as a major sensor for the amino acid levels has been demonstrated in experiments in cultured cells particularly using siRNA-based downregulation of V-ATPase subunits. As our own data have shown, the situation appears more complex in vivo since a liver-specific, induced deletion of the V-ATPase complex surprisingly elevates mTORC1 activity while, at the same time, massively increasing the number of autophagic vacuoles (Kissing et al., 2017). These observations indicate that at least in certain cell types pathways next to the control of mTORC1 by lysosome-resident V-ATPase must be relevant to maintain mTORC1 activity and to regulate autophagic flux.
V-ATPase: more than a proton pump In baker’s yeast, knockout and mutational studies revealed an acidification-independent requirement for the V-ATPase VO sector in the process of homotypic vacuole fusion (Peters et al., 2001, Strasser et al., 2011). It was proposed that conformational changes within the VO sector following the formation of trans-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) complexes would promote fusion. This rearrangement would allow acyl chains from the lipid bilayer to invade a gap in the VO sector promoting the curvature of the membrane and allowing the formation of a fusion pore (Clare et al., 2006, Strasser et al., 2011). Follow-up studies in higher eukaryotes confirmed this new function of V-ATPase in membrane fusion. For example the rat VO subunit c modulates neurotransmitter release (Di Giovanni et al., 2010). Similarly, mutations of the VO a-subunit are correlated with defective secretion of Hedgehog-related proteins in Caenorhabditis elegans (Liegeois et al., 2006) and lead to impaired secretion of insulin from mouse pancreatic islets cells (Sun-Wada et al., 2006). The fly homologue of the VO subunit a1 regulates synaptic vesicle release in association with SNARE complexes (Hiesinger et al., 2005, Wang et al., 2014). In fact, photo-inactivation of the VO subunit a1 in chromaffin cells helped to identify the V-ATPase VO sector as a sensor for intra-granular pH, which again regulates exocytosis (Poea-Guyon et al., 2013). A proton pumping-independent and general role of V-ATPases in membrane fusion was challenged by observations in yeast where impaired vacuole fusion was attributed to a lack of V-ATPase-mediated vesicle acidification rather than a lack of the pump itself (Coonrod et al., 2013). However, more recent studies describe a requirement for the presence of the complex during yeast membrane fusion in vitro and in vivo (Desfougeres et al., 2016, Sreelatha et al., 2015).