International Journal of Cell Biology disorders such as Huntington’s disease. Here, fragments of the disease-related protein containing the polyQ tract initiate aggregation and toxicity, which can be mimicked by expressing the expanded polyQ sequence as a peptide [42]. Apparently, not all peptides are eﬃciently degraded by peptidases, which led to our recently published study where we examined potential alternative degradation machineries when peptidases would fail in degrading protein fragments [43]. In this study, we introduced peptidase-resistant pep- tides into living cells and observed a perinuclear accumula- tion of these peptides in time. Surprisingly, these structures did not represent aggregates or inclusion bodies as observed previously for aggregation-prone protein fragments, as no UPS components or chaperones were recruited. Although initially present in the nucleus and cytoplasm, the peptides were eﬃciently targeted to lysosomes within a few hours upon introduction into cells, and subsequently degraded. Our results indicate, therefore, that similar to the described increase in autophagy upon proteasome impairment, a backup mechanism exists for small protein fragments that show peptidase resistance. Intriguingly, this mechanism was very eﬃcient for peptides of the average size of proteasomal products (6–9 amino acids), but far less for extended peptides over 25–30 amino acids which remained cytoplasmic for prolonged periods [43]. Similar to expanded polyQ peptides of disease-related lengths, these expanded peptidase-resistant peptides were more resistant to clearance by lysosomes suggesting that this pathway is particularly eﬃcient for small peptides generated by the proteasome. It is tempting to speculate that this mechanism evolved as a backup to peptidases in the clearance of proteasome- derived peptides and emphasizes the need to identify the involved proteins. Using correlative microscopy, we mainly observed double-membrane vesicles that contained peptides and that colocalized with LC3. The colocalization increased when we used Baﬁlomycin A1 to impair maturation into autolysosomes. In contrast, we could prevent colocalization of LC3 with the macroautophagy inhibitor 3-MA, suggesting that the macroautophagic pathway took over the clearance of these peptides. Unexpectedly, inhibition of macroautophagy by inhibitors such as 3-MA or knockdown of Atg5 prevented recruitment of LC3 but did not aﬀect the traﬃcking of these peptides into lysosomes or their subsequent degradation. Apparently, LC3 was recruited during the traﬃcking of peptides towards lysosomes yet was not essential. Similar to the knockdown of the various LC3 isoforms (LC3A-C), knockdown of the Atg8-related GABARAP proteins, that can interact with autophagosomes, did not aﬀect the targeting of peptides towards lysosomes [44, 45]. As knockdown of Atg5 or WIPI-1 did not aﬀect the traﬃcking and subsequent degradation of peptides in lysosomes, we concluded that these peptides entered lysosomes via a pathway diﬀerent from macroautophagy. CMA is also unlikely to contribute to this pathway as the peptides lack a CMA motif and peptides composed of D-amino acids, which are unable to bind chaperones like Hsc70, were also traﬃcking via this pathway. Finally, we also examined endosomal microautophagy, a process that delivers soluble cytosolic material to vesicles of late endosomes or multivesicular bodies (MVBs) [46, 47]. 3 Although accumulated peptides colocalized with internalized MHC class II molecules which may lead to so-called cross- presentation to the immune system (unpublished observa- tion), knockdown of the sorting complexes required for transport (ESCRTs) I and III showed no eﬀect on peptide accumulation in lysosomes. As no recruitment of ESCRT regulators towards accumulated peptides was observed, this indicates that the endosomal microautophagy pathway is not involved in the traﬃcking and clearance of the peptidase- resistant peptides. The accumulation and subsequent lysosomal degrada- tion of cytoplasmic proteins independent of known auto- phagy pathways have been previously observed in several studies (as described below), although in each case diﬀer- ences in sensitivity to autophagy inhibitors and the involve- ment of various Atg proteins were reported. Interestingly, in a study using Green Fluorescent Protein (GFP) like ﬂuorophores, a pathway reminiscent of that we observed for the peptidase-resistant peptides was observed [40]. Various GFP-like ﬂuorophores have been shown to form dimers, tetramers, or even larger complexes. Upon expression, these ﬂuorescent proteins formed cytoplasmic ﬂuorescent puncta that resembled lysosomes, similar as observed for the pep- tidase-resistant peptides [48]. However, the accumulating ﬂuorophore proteins including monomeric RFP1 (mRFP1) showed resistance to lysosomal degradation and retain ﬂu- orescence, in contrast to the peptides. Traﬃcking of the GFP-like proteins and the peptidase-resistant peptides was not aﬀected in Atg5-deﬁcient mouse embryonic ﬁbroblasts, suggesting that they may be targeted to lysosomes by a similar pathway (although no other macroautophagy markers were examined for the ﬂuorescent proteins). So is the constitutive macroautophagy-independent targeting of cytoplasmic pro- teins and peptides to autolysosomes restricted to introduced peptides and GFP-like ﬂuorophores? At least two alternative autophagy pathways have been described: an Atg5/Atg7-independent pathway and the so- called noncanonical autophagy pathway, which is inde- pendent of Beclin 1 (Table 1). The Atg5/Atg7-independent autophagic pathway was recently discovered in mouse embryonic ﬁbroblasts (MEF) lacking Atg5 and Atg7 that were treated with the cytotoxic stressor etoposide, which caused an equivalent appearance of autophagic vacuoles when compared to wild-type cells [49]. Moreover, auto- phagic vacuoles were also found in starved Atg5-/- cells. The Atg5/Atg7-independent form of autophagy does not involve the lipidated conjugate LC3-II, which is membrane associ- ated. Interestingly, equivalent numbers of LC3-positive and LC3-negative autophagosomes were observed in etoposide- treated wild-type cells, suggesting that conventional and alternative autophagic pathway occur at the same time. The proteins Atg5, Atg7, and LC3, which are important in the ubiquitin-like conjugation system for the autophago- some elongation, are not involved in this alternative form of autophagy. However, silencing of Beclin 1 and Vps34 decreased the amount of autophagosomes, indicating that the PI3K complex, which acts upstream of initiation of autophagosome formation, is still required in etoposide- or starvation-induced autophagy in Atg5-/- cells. Accordingly,