Lysosomes

Lysosomes were first discovered and described by Christian de Duve as the main degradative organelles of eukaryotic cells (de Duve et al. 1955). These membrane-bound organelles, which are in average 0.1-2 µm in diameter (Novikoff et al. 1956), are enclosed by a lipid-bilayer and have an acidic lumen (pH 4.5-5). The acidification, which is regulated by a vacuolar H⁺-ATPase that pumps protons into the lumen (Finbow and Harrison 1997), maintains a high activity of at least 60 different soluble lysosomal hydrolases (Lübke et al. 2009) that are involved in the breakdown of polysaccharides, proteins, lipids and other macromolecules (Luzio et al. 2007). Deficiencies in the lysosomal enzymes can lead to numerous lysosomal storage diseases (Ballabio and Gieselmann 2009).

More than 100 highly glycosylated membrane proteins are directly involved in lysosomal functions by maintaining the lysosomal membrane integrity, regulating the transport of metabolites across the lysosomal membrane as well as modulating lysosomal motility and fusion capability (Saftig 2005; Huynh et al.

2007; Ruivo et al. 2009; Schwake et al. 2013). Along with lysosomal membrane proteins, lipids protect the integrity of the membrane to prevent the release of hydrolases into the cytosol, which may lead to lysosomal-dependent cell death (Wang et al. 2018). Lysosomes are also involved in biological and physiological functions such as plasma membrane repair (Pu et al. 2016), production of inflammatory cytokines (Ge et al. 2015) and osteoclastogenesis (Erkhembaatar et al. 2017).

1.2.1 Lysosomal adaptation in response to nutrient status

Lysosomes are dynamic organelles, which can adapt to alterations in nutrient availability via changes in their size, number, enzyme activity and positioning (Reviewed by Settembre and Ballabio 2014). Apart from the lysosomal luminal enzymes, the solute transporters and the motility proteins, the lysosome has a

set of membrane-associated complexes that can respond to the changes in nutrient status, primarily via the master regulator of cell growth and metabolism, the mechanistic target of rapamycin (mTOR) as part of mTOR complex 1 (mTORC1) (Sancak et al. 2010). In addition to the 289-kDa serine/threonine protein kinase, the signaling complex consists of four components: the regulatory-associated protein of mTOR (RAPTOR); the proline-rich AKT substrate 40 kDa (PRAS40); the DEP-domain-containing mTOR-interacting protein (DEPTOR); and the mammalian lethal with Sec13 protein 8 (mLST8) (Sengupta et al. 2010b) as shown in figure 1.3. Moreover, mTOR is a catalytic subunit of a distinct mTORC2 complex (Reviewed by Liu and Sabatini 2020).

Figure 1.3: Components of the mTORC1 complex

Cartoon illustrating the structure of mTORC1 complex. mTORC1 is composed of five know protein components: mTOR, RAPTOR, mLST8, PRAS40 and DEPTOR.

In the presence of nutrients, mTORC1 is localized on the lysosomal surface and can sense the availability of the nutrient components like amino acids. During starvation, the inactive complex dissociates into the cytosol as part of a well-known adaptive mechanism to nutrient deprivation (Kim et al. 2013). Inactivation of mTORC1 enables autophagy initiation and nuclear translocation of the transcription factor EB (TFEB) to activate lysosomal biogenesis (Settembre et al.

2012; Roczniak-Ferguson et al. 2012). Lysosomes are relocated in order to fuse with the newly formed autophagosomes. This process is mediated by several autophagy-related (ATG) proteins such as phosphatidylethanolamine-conjugated LC3-II, which is associated with the inner and outer membrane of the autophagosome throughout the autophagy process (Korolchuk and Rubinsztein 2011; Ktistakis and Tooze 2016; Nguyen et al. 2016). Higher acidification in the lysosomal lumen leads to an increase in the degradation of macromolecules and the cytosolic release of amino acids to enable the reactivation of mTORC1 after

Introduction 8

a prolonged starvation (Yu et al. 2010). The number of lysosomes is finally adapted via lysosomal biogenesis and autophagic lysosome reformation (ALR) (Shen and Mizushima 2014).

1.2.2 The molecular mechanisms of nutrient signaling at the lysosome Lysosomes play a crucial role as a cellular signaling hub, regulating different metabolic pathways like cell growth (Sancak et al. 2008). In nutrient-rich conditions, mTORC1 activation and lysosomal positioning are controlled by several proteins mainly the arginine-regulated transporter SLC38A9 (Wang et al.

2015) and the lysosomal nutrient sensing machinery (LYNUS), consisting of vacuolar H⁺-ATPase, Rag GTPases, Rag GAPs and Ragulator complex (Settembre et al. 2013; Wolfson and Sabatini 2017) (Figure 1.4).

SLC38A9, a positive regulator of mTORC1, senses the arginine in the lysosome and activates the Ragulator complex. The active pentameric complex consisting of LAMTOR1-5 functions as guanine nucleotide exchange factor (GEF) for Rag GTPases heterodimers converting them into their active forms with GTP bound-RagA/B and GDP bound-RagC/D (Sancak et al. 2010; Bar-Peled et al. 2012).

Active Rags then recruit and bind mTORC1 at the lysosomal surface (Figure 1.4).

Another essential activator of mTORC1 is the small GTPase Rheb, ras homolog enriched in brain, which directly activates it through a strong interaction (Saito et al. 2005; Buerger et al. 2006). This interaction is negatively regulated by the tuberous sclerosis complex (TSC) consisting of TSC1, TSC2 and TBC1 domain family member 7 (TBC1D7) (Dibble et al. 2012). TSC’s strong GTPase activating protein (GAP) can convert Rheb into its GDP-bound inactive form and thus inhibits it. As regulators of Rag GTPases, Folliculin and its bound proteins FNIP1/2 have a GAP activity against RagC/D (Tsun et al. 2013). At the same time, the GAP activity towards the Rags 1 complex (GATOR1) consisting of DEPDC5, NPRL2 and NPRL3, as well as its partner GATOR2 complex consisting of WDR59, WDR24, MIOS, SEH1L and SEC13 can regulate RagA/B by their GAP activity (Bar-Peled et al. 2013).

Figure 1.4: mTORC1 upstream nutrient signaling pathway

Schematic describing the nutrient sensing pathway upstream of mTORC1 and its key molecular components (Wolfson and Sabatini 2017).

GATOR1 and GATOR2 have recently being identified to be recruited to the lysosomal surface by the KICSTOR complex which is composed of KPTN, ITFG2, C12orf66 and SZT2 (Figure 1.4). The vacuolar H⁺-ATPase pump is suggested to sense the amino acids in the lysosome and send a signal to Rag GTPases via the Ragulator complex (Figure 1.4) (Zoncu et al. 2011).

1.2.3 Downstream targets and functions of lysosomal mTORC1

Several anabolic and catabolic reactions need to be regulated for normal cell growth and division (Kuo et al. 1992). When nutrient and energy are abundant, activated mTORC1 triggers a variety of cellular processes such as protein, lipid and nucleotide synthesis, along with suppressing autophagy and thus lysosome biogenesis by phosphorylation of distinct substrates (Figure 1.5) (Reviewed by Liu and Sabatini 2020).

mTORC1 stimulates protein synthesis by phosphorylating p70S6 Kinase 1 (S6K1) and eIF4E Binding Protein 1 (4EBP1) (Figure 1.5). Direct phosphorylation of S6K1 promotes mRNA translation initiation and leads to the activation of several substrates. S6K1 phosphorylates the S6 protein, a ribosomal 40S subunit that is claimed to be involved in the transcription of ribosomal genes (Chauvin et

Introduction 10

al. 2014). 4EBP1 inhibits translation by binding the eukaryotic translation initiation factor 4E (eIF4E) and thus prevents the assembly of eIF4F complex. mTORC1 phosphorylates 4EBP1 and dissociates it from eIF4E and allows a 5′ cap-dependent mRNA translation (Pause et al. 1994; Brunn et al. 1997; Hara et al.

1997; Gingras et al. 1999).

Figure 1.5: mTORC1 downstream signaling targets

Schematic representation of mTORC1 activation and the initiation of anabolic processes that stimulate the production of proteins, lipids and nucleotides as well as the inhibition of catabolic programs such as autophagy.

For a balanced cell membrane expansion, mTORC1 promotes lipid synthesis through the transcription factors sterol regulatory element binding protein 1/2 (SREBP1/2) (Porstmann et al. 2008), which is activated by S6K-dependent mechanisms or lipin-1. Lipin-1 gets inhibited by mTORC1 phosphorylation (Düvel et al. 2010; Peterson et al. 2011).

mTORC1 also regulates DNA replication and nucleotide synthesis by stimulating ATF4 transcription factor and the mitochondrial tetrahydrofolate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) associated with purine synthesis (Ben-Sahra et al. 2016).

Besides the anabolic reactions, mTORC1 controls cell growth by suppressing catabolic processes such as autophagy. In the presence of nutrients, mTORC1 inhibits autophagy via the phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) (Figure 1.5). As a result, phosphorylated ULK1 is prevented from

activating AMPK and thus the initiation of autophagosome formation is inhibited (Zhao and Goldberg 2016).