2 the endoplasmatic reticulum (ER) [14, 15]. However, alter- native sources for the autophagosomal membrane have been proposed, including the Golgi apparatus, and therefore the origin of the phagophore membrane still remains unresolved [16, 17]. 2. Macroautophagy Macroautophagy is a multistep process controlled by pro- teins termed autophagy-related (Atg) proteins [18]. The formation of the phagophore requires the class-III-phos- phatidylinositol 3-kinase (PI3K) Vps34 that forms a com- plex with Beclin 1 (the mammalian orthologue of yeast Atg6). Inhibitors of Vps34 such as methyladenine (3-MA) or wortmannin can be used to inhibit macroautophagy since they prevent autophagosome nucleation [19–22]. The elongation of the autophagosomal membrane is dependent on two ubiquitin-like conjugation systems [23]. Atg5-Atg12 controls autophagy, where Atg12 is conjugated to Atg5 in a step that requires Atg7 (ubiquitin-activating-enzyme (E1)- like) and Atg10 (ubiquitin-conjugating-enzyme (E2)-like). The Atg5-Atg12 conjugation depends on Vps34 activity and is localized onto the phagophore where it dissociates upon formation of the autophagosome. Atg5-Atg12 forms a complex with Atg16L that modulates the next process, the ubiquitin-like conjugation of LC3-I (mammalian ortho- logue of Atg8). The protein LC3 is proteolytic activated by Atg4, which cleaves the C-terminus of LC3, thereby generating a cytosolic LC3-I, which subsequently conjugates with phosphatidylethanolamine (PE) to form membrane- associated LC3-II [24]. This process requires Atg7 and Atg3, and the Atg16L complex modulates the LC3-I lip- idation by acting like an E3-like enzyme [25]. Although the Atg5-Atg12 conjugation dissociates upon completion of the autophagosome formation, LC3-II persists with the autophagosomal membrane even after fusion with a lyso- somes and is regarded as a key marker for autophagosomes. Atg4 is also involved in the deconjugation reaction of LC3-II, as Atg4 delipidates LC3-II and removes it from the autophagosomal membrane [24, 26]. A pathway that negatively regulates macroautophagy is controlled by mTOR (mammalian target of rapamycin). mTOR activity is inhib- ited under starvation conditions, which activates starvation- induced macroautophagy. Recently, two new key regulators of macroautophagy, named NIX and DOR, which directly interact with the autophagosome-membrane-associated pro- tein LC3, were identiﬁed [27]. Nix, a Bcl2-related protein localized the outer mitochondrial membrane, has a function as an adaptor protein and recruits autophagic components to mitochondria via its WXXL-like domain facing the cytoplasm [28–30]. NIX is upregulated during erythroid diﬀerentiation where a lack of mitochondria is achieved by mitophagy [27, 31, 32]. Interestingly, NIX-deﬁcient mice show remaining mitochondria in matured red blood cells suggesting that NIX is a selective autophagy receptor that mediates mitochondrial clearance, as it directly binds LC3, but it may also target mitochondria for degradation in an LC3-independent manner [27, 33, 34]. Intriguingly, in International Journal of Cell Biology the same issue of EMBO reports, another new autophagy- related protein was reported. Mauvezin et al. identiﬁed the nuclear cofactor of thyroid hormone receptors, termed DOR (diabetes- and obesity-regulated gene), as a new player of macroautophagy [35]. Stress-induced macroautophagy by starvation or rapamycin leads to release of DOR from the nucleus in DOR-transfected HeLa cells. This relocal- ization was not observed in the absence of cellular stress, indicating that cellular stress is essential to trigger DOR recruitment to the cytoplasm. DOR is associated with early autophagosomes via interaction with LC3 and GATE16 but does not colocalize with autolysosomes suggesting that DOR has a regulatory role in recruiting substrates for autophagic clearance. In addition, DOR-transfected HeLa cells show increased turnover of proteins and elevated numbers of autophagosomes compared to untreated cells. It has yet to be discovered which role DOR is playing, as it may be involved in targeting proteins to autophagy or in the formation and nucleation of the autophagosome. Whether DOR activation aﬀects autophagy-induced alterations in cell survival remains to be established. Macroautophagy was originally described to target intra- cellular organelles such as mitochondria and big protein complexes, but over the years it became clear that also most long-lived proteins are degraded via autophagic pathways. In contrast, the other main degradation machinery in the cell, the UPS, degrades mainly soluble short-lived and misfolded proteins that are targeted to the proteasome following ubiq- uitination (using a series of E1-E2-E3 enzymes to speciﬁcally target proteins for destruction). The proteasome is present in both the cytoplasm and the nucleus and can unfold and degrade single proteins into small peptide fragments that are subsequently recycled by peptidases. Interestingly, impair- ment of the proteasome leads to an increase in macroau- tophagy, indicating that macroautophagy can target accu- mulating ubiquitinated proteasomal clients when required [36–39]. In contrast, impairment of macroautophagy does not lead to increased proteasome activity. Inhibition of macroautophagy does not aﬀect the catalytic activity of the proteasome but results in the accumulation of the macroau- tophagy cargo receptor p62 (also termed SQSTM1) which competes with the proteasome for ubiquitinated substrates. Indeed, silencing of p62 increases the amount of UPS clients, whereas overexpression of p62 inhibits degradation of the proteasomal substrates p53 and UbG76V-GFP [40, 41]. As p62 links ubiquitinated proteins via its ubiquitin-associated (UBA) domain to the autophagic protein LC3-II and is itself degraded in the process, inhibition of macroautophagy leads to p62 accumulation which will compete and frustrate other ubiquitin-binding proteins that participate in proteasome- mediated degradation. 3. Alternative Autophagic Pathways Failure of the UPS or autophagic pathways to eﬃciently clear proteins leads to the accumulation and subsequent aggrega- tion of these proteins, which is a hallmark of various neu- rodegenerative disorders including polyglutamine (polyQ)